Nucleic acids encoding kringle 1-5 region fragments of plasminogen

ABSTRACT

Fragments of an endothelial cell proliferation inhibitor and method of use therefor are provided. The endothelial proliferation inhibitor is a protein derived from plasminogen, or more specifically is an angiostatin fragment. The angiostatin fragments generally correspond to kringle structures occurring within the endothelial cell proliferation inhibitor. The endothelial cell inhibiting activity of these fragments provides a means for inhibiting angiogenesis of tumors and for treating angiogenic-mediated disease.

FIELD OF THE INVENTION

[0001] The present invention relates to endothelial inhibitors, calledangiostatin, which reversibly inhibit proliferation of endothelialcells. More particularly, the present invention relates to angiostatinproteins that can be isolated from body fluids such as blood or urine,or can be synthesized by recombinant, enzymatic or chemical methods. Theangiostatin is capable of inhibiting angiogenesis related diseases andmodulating angiogenic processes. In addition, the present inventionrelates to diagnostic assays and kits for angiostatin measurement, tohistochemical kits for localization of angiostatin, to DNA sequencescoding for angiostatin and molecular probes to monitor angiostatinbiosynthesis, to antibodies that are specific for the angiostatin, tothe development of protein agonists and antagonists to the angiostatinreceptor, to anti-angiostatin receptor-specific antibody agonists andantagonists, and to cytotoxic agents linked to angiostatin proteins.

BACKGROUND OF THE INVENTION

[0002] As used herein, the term “angiogenesis” means the generation ofnew blood vessels into a tissue or organ. Under normal physiologicalconditions, humans or animals undergo angiogenesis only in very specificrestricted situations. For example, angiogenesis is normally observed inwound healing, fetal and embryonal development and formation of thecorpus luteum, endometrium and placenta. The term “endothelium” means athin layer of flat epithelial cells that lines serous cavities, lymphvessels, and blood vessels.

[0003] Both controlled and uncontrolled angiogenesis are thought toproceed in a similar manner. Endothelial cells and pericytes, surroundedby a basement membrane, form capillary blood vessels. Angiogenesisbegins with the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating the new blood vessel.

[0004] Persistent, unregulated angiogenesis occurs in a multiplicity ofdisease states, tumor metastasis and abnormal growth by endothelialcells and supports the pathological damage seen in these conditions. Thediverse pathological disease states in which unregulated angiogenesis ispresent have been grouped together as angiogenic dependent or angiogenicassociated diseases.

[0005] The hypothesis that tumor growth is angiogenesis-dependent wasfirst proposed in 1971. (Folkman J., Tumor angiogenesis: Therapeuticimplications., N. Engl. Jour. Med. 285:1182 1186, 1971) In its simplestterms it states: “Once tumor ‘take’ has occurred, every increase intumor cell population must be preceded by an increase in new capillariesconverging on the tumor.” Tumor ‘take’ is currently understood toindicate a prevascular phase of tumor growth in which a population oftumor cells occupying a few cubic millimeters volume and not exceeding afew million cells, can survive on existing host microvessels. Expansionof tumor volume beyond this phase requires the induction of newcapillary blood vessels. For example, pulmonary micrometastases in theearly prevascular phase in mice would be undetectable except by highpower microscopy on histological sections.

[0006] Examples of the indirect evidence which support this conceptinclude:

[0007] (1) The growth rate of tumors implanted in subcutaneoustransparent chambers in mice is slow and linear beforeneovascularization, and rapid and nearly exponential afterneovascularization. (Algire G H, et al. Vascular reactions of normal andmalignant tumors in vivo. I. Vascular reactions of mice to wounds and tonormal and neoplastic transplants. J. Natl. Cancer Inst. 6:73-85, 1945)

[0008] (2) Tumors grown in isolated perfused organs where blood vesselsdo not proliferate are limited to 1-2 mm³ but expand rapidly to >1000times this volume when they are transplanted to mice and becomeneovascularized. (Folkman J, et al., Tumor behavior in isolated perfusedorgans: In vitro growth and metastasis of biopsy material in rabbitthyroid and canine intestinal segments. Annals of Surgery 164:491-502,1966)

[0009] (3) Tumor growth in the avascular cornea proceeds slowly and at alinear rate, but switches to exponential growth afterneovascularization. (Gimbrone, M. A., Jr. et al., Tumor growth andneovascularization: An experimental model using the rabbit cornea. J.Natl. Cancer Institute 52:41-427, 1974)

[0010] (4) Tumors suspended in the aqueous fluid of the anterior chamberof the rabbit eye, remain viable, avascular and limited in size to <1mm³. Once they are implanted on the iris vascular bed, they becomeneovascularized and grow rapidly, reaching 16,000 times their originalvolume within 2 weeks. (Gimbrone M A Jr., et al., Tumor dormancy in vivoby prevention of neovascularization. J. Exp. Med. 136:261-276)

[0011] (5) When tumors are implanted on the chick embryo chorioallantoicmembrane, they grow slowly during an avascular phase of >72 hours, butdo not exceed a mean diameter of 0.93+0.29 mm. Rapid tumor expansionoccurs within 24 hours after the onset of neovascularization, and by day7 these vascularized tumors reach a mean diameter of 8.0+2.5 mm.(Knighton D., Avascular and vascular phases of tumor growth in the chickembryo. British J. Cancer, 35:347-356, 1977)

[0012] (6) Vascular casts of metastases in the rabbit liver revealheterogeneity in size of the metastases, but show a relatively uniformcut-off point for the size at which vascularization is present. Tumorsare generally avascular up to 1 mm in diameter, but are neovascularizedbeyond that diameter. (Lien W., et al., The blood supply of experimentalliver metastases. II. A microcirculatory study of normal and tumorvessels of the liver with the use of perfused silicone rubber. Surgery68:334-340, 1970)

[0013] (7) In transgenic mice which develop carcinomas in the beta cellsof the pancreatic islets, pre-vascular hyperplastic islets are limitedin size to <1 mm. At 6-7 weeks of age, 4-10% of the islets becomeneovascularized, and from these islets arise large vascularized tumorsof more than 1000 times the volume of the pre-vascular islets. (FolkmanJ, et al., Induction of angiogenesis during the transition fromhyperplasia to neoplasia. Nature 339:58-61, 1989)

[0014] (8) A specific antibody against VEGF (vascular endothelial growthfactor) reduces microvessel density and causes “significant or dramatic”inhibition of growth of three human tumors which rely on VEGF as theirsole mediator of angiogenesis (in nude mice). The antibody does notinhibit growth of the tumor cells in vitro. (Kim K J, et al., Inhibitionof vascular endothelial growth factor-induced angiogenesis suppressestumor growth in vivo. Nature 362:841-844, 1993)

[0015] (9) Anti-bFGF monoclonal antibody causes 70% inhibition of growthof a mouse tumor which is dependent upon secretion of bFGF as its onlymediator of angiogenesis. The antibody does not inhibit growth of thetumor cells in vitro. (Hori A, et al., Suppression of solid tumor growthby immunoneutralizing monoclonal antibody against human basic fibroblastgrowth factor. Cancer Research, 51:6180-6184, 1991)

[0016] (10) Intraperitoneal injection of bFGF enhances growth of aprimary tumor and its metastases by stimulating growth of capillaryendothelial cells in the tumor. The tumor cells themselves lackreceptors for bFGF, and bFGF is not a mitogen for the tumors cells invitro. (Gross J L, et al. Modulation of solid tumor growth in vivo bybFGF. Proc. Amer. Assoc. Canc. Res. 31:79, 1990)

[0017] (11) A specific angiogenesis inhibitor (AGM-1470) inhibits tumorgrowth and metastases in vivo, but is much less active in inhibitingtumor cell proliferation in vitro. It inhibits vascular endothelial cellproliferation half-maximally at 4 logs lower concentration than itinhibits tumor cell proliferation. (Ingber D, et al., Angioinhibins:Synthetic analogues of fumagillin which inhibit angiogenesis andsuppress tumor growth. Nature, 48:555-557, 1990). There is also indirectclinical evidence that tumor growth is angiogenesis dependent.

[0018] (12) Human retinoblastomas that are metastatic to the vitreousdevelop into avascular spheroids which are restricted to less than 1 mm³despite the fact that they are viable and incorporate ³H-thymidine (whenremoved from an enucleated eye and analyzed in vitro).

[0019] (13) Carcinoma of the ovary metastasizes to the peritonealmembrane as tiny avascular white seeds (1-3 mm³). These implants rarelygrow larger until one or more of them becomes neovascularized.

[0020] (14) Intensity of neovascularization in breast cancer (Weidner N,et al., Tumor angiogenesis correlates with metastasis in invasive breastcarcinoma. N. Engl. J. Med. 324:1-8, 1991, and Weidner N, et al., Tumorangiogenesis: A new significant and independent prognostic indicator inearly-stage breast carcinoma, J Natl. Cancer Inst. 84:1875-1887, 1992)and in prostate cancer (Weidner N, Carroll P R, Flax J, Blumenfeld W,Folkman J. Tumor angiogenesis correlates with metastasis in invasiveprostate carcinoma. American Journal of Pathology, 143(2):401-409, 1993)correlates highly with risk of future metastasis.

[0021] (15) Metastasis from human cutaneous melanoma is rare prior toneovascularization. The onset of neovascularization leads to increasedthickness of the lesion and an increasing risk of metastasis.(Srivastava A, et al., The prognostic significance of tumor vascularityin intermediate thickness (0.76-4.0 mm thick) skin melanoma. Amer. J.Pathol. 133:419-423, 1988)

[0022] (16) In bladder cancer, the urinary level of an angiogenicprotein, bFGF, is a more sensitive indicator of status and extent ofdisease than is cytology. (Nguyen M, et al., Elevated levels of anangiogenic protein, basic fibroblast growth factor, in urine of bladdercancer patients. J. Natl. Cancer Inst. 85:241-242, 1993)

[0023] Thus, it is clear that angiogenesis plays a major role in themetastasis of a cancer. If this angiogenic activity could be repressedor eliminated, then the tumor, although present, would not grow. In thedisease state, prevention of angiogenesis could avert the damage causedby the invasion of the new microvascular system. Therapies directed atcontrol of the angiogenic processes could lead to the abrogation ormitigation of these diseases.

[0024] What is needed therefore is a composition and method which caninhibit the unwanted growth of blood vessels, especially into tumors.Also needed is a method for detecting, measuring, and localizing thecomposition. The composition should be able to overcome the activity ofendogenous growth factors in premetastatic tumors and prevent theformation of the capillaries in the tumors thereby inhibiting the growthof the tumors. The composition, fragments of the composition, andantibodies specific to the composition, should also be able to modulatethe formation of capillaries in other angiogenic processes, such aswound healing and reproduction. The composition and method forinhibiting angiogenesis should preferably be non-toxic and produce fewside effects. Also needed is a method for detecting, measuring, andlocalizing the binding sites for the composition as well as sites ofbiosynthesis of the composition. The composition and fragments of thecomposition should be capable of being conjugated to other molecules forboth radioactive and non-radioactive labeling purposes

SUMMARY OF THE INVENTION

[0025] In accordance with the present invention, compositions andmethods are provided that are effective for modulating angiogenesis, andinhibiting unwanted angiogenesis, especially angiogenesis related totumor growth. The present invention includes a protein, which has beennamed “angiostatin”, defined by its ability to overcome the angiogenicactivity of endogenous growth factors such as bFGF, in vitro, and by itamino acid sequence homology and structural similarity to an internalportion of plasminogen beginning at approximately plasminogen amino acid98. Angiostatin comprises a protein having a molecular weight of betweenapproximately 38 kilodaltons and 45 kilodaltons as determined byreducing polyacrylamide gel electrophoresis and having an amino acidsequence substantially similar to that of a fragment of murineplasminogen beginning at amino acid number 98 of an intact murineplasminogen molecule (SEQ ID NO:2).

[0026] The amino acid sequence of angiostatin varies slightly betweenspecies. For example, in human angiostatin the amino acid sequence issubstantially similar to the sequence of the above described murineplasminogen fragment, although an active human angiostatin sequence maystart at either amino acid number 97 or 99 of an intact humanplasminogen amino acid sequence. Further, fragments of human plasminogenhas similar anti-angiogenic activity as shown in a mouse tumor model. Itis to be understood that the number of amino acids in the activeangiostatin molecule may vary and all amino acid sequences that haveendothelial inhibiting activity are contemplated as being included inthe present invention.

[0027] The present invention provides methods and compositions fortreating diseases and processes mediated by undesired and uncontrolledangiogenesis by administering to a human or animal a compositioncomprising a substantially purified angiostatin or angiostatinderivative in a dosage sufficient to inhibit angiogenesis. The presentinvention is particularly useful for treating, or for repressing thegrowth of, tumors. Administration of angiostatin to a human or animalwith prevascularized metastasized tumors will prevent the growth orexpansion of those tumors.

[0028] The present invention also encompasses DNA sequences encodingangiostatin, expression vectors containing DNA sequences encodingangiostatin, and cells containing one or more expression vectorscontaining DNA sequences encoding angiostatin. The present inventionfurther encompasses gene therapy methods whereby DNA sequences encodingangiostatin are introduced into a patient to modify in vivo angiostatinlevels.

[0029] The present invention also includes diagnostic methods and kitsfor detection and measurement of angiostatin in biological fluids andtissues, and for localization of angiostatin in tissues and cells. Thediagnostic method and kit can be in any configuration well known tothose of ordinary skill in the art. The present invention also includesantibodies specific for the angiostatin molecule and portions thereof,and antibodies that inhibit the binding of antibodies specific for theangiostatin. These antibodies can be polyclonal antibodies or monoclonalantibodies. The antibodies specific for the angiostatin can be used indiagnostic kits to detect the presence and quantity of angiostatin whichis diagnostic or prognostic for the occurrence or recurrence of canceror other disease mediated by angiogenesis. Antibodies specific forangiostatin may also be administered to a human or animal to passivelyimmunize the human or animal against angiostatin, thereby reducingangiogenic inhibition.

[0030] The present invention also includes diagnostic methods and kitsfor detecting the presence and quantity of antibodies that bindangiostatin in body fluids. The diagnostic method and kit can be in anyconfiguration well known to those of ordinary skill in the art.

[0031] The present invention also includes anti-angiostatinreceptor-specific antibodies that bind to the angiostatin receptor andtransmit the appropriate signal to the cell and act as agonists orantagonists.

[0032] The present invention also includes angiostatin protein fragmentsand analogs that can be labeled isotopically or with other molecules orproteins for use in the detection and visualization of angiostatinbinding sites with techniques, including, but not limited to, positronemission tomography, autoradiography, flow cytometry, radioreceptorbinding assays, and immunohistochemistry.

[0033] These angiostatin proteins and analogs also act as agonists andantagonists at the angiostatin receptor, thereby enhancing or blockingthe biological activity of angiostatin. Such proteins are used in theisolation of the angiostatin receptor.

[0034] The present invention also includes angiostatin, angiostatinfragments, angiostatin antisera, or angiostatin receptor agonists andangiostatin receptor antagonists linked to cytotoxic agents fortherapeutic and research applications. Still further, angiostatin,angiostatin fragments, angiostatin antisera, angiostatin receptoragonists and angiostatin receptor antagonists are combined withpharmaceutically acceptable excipients, and optionally sustained-releasecompounds or compositions, such as biodegradable polymers, to formtherapeutic compositions.

[0035] The present invention includes molecular probes for theribonucleic acid and deoxyribonucleic acid involved in transcription andtranslation of angiostatin. These molecular probes provide means todetect and measure angiostatin biosynthesis in tissues and cells.

[0036] Accordingly, it is an object of the present invention to providea composition comprising an angiostatin.

[0037] It is another object of the present invention to provide a methodof treating diseases and processes that are mediated by angiogenesis.

[0038] It is yet another object of the present invention to provide adiagnostic or prognostic method and kit for detecting the presence andamount of angiostatin in a body fluid or tissue.

[0039] It is yet another object of the present invention to provide amethod and composition for treating diseases and processes that aremediated by angiogenesis including, but not limited to, hemangioma,solid tumors, blood borne tumors, leukemia, metastasis, telangiectasia,psoriasis, scleroderma, pyogenic granuloma, myocardial angiogenesis,Crohn's disease, plaque neovascularization, coronary collaterals,cerebral collaterals, arteriovenous malformations, ischemic limbangiogenesis, corneal diseases, rubeosis, neovascular glaucoma, diabeticretinopathy, retrolental fibroplasia, arthritis, diabeticneovascularization, macular degeneration, wound healing, peptic ulcer,Helicobacter related diseases, fractures, keloids, vasculogenesis,hematopoiesis, ovulation, menstruation, placentation, and cat scratchfever.

[0040] It is another object of the present invention to provide acomposition for treating or repressing the growth of a cancer.

[0041] It is an object of the present invention to provide compoundsthat modulate or mimic the production or activity of enzymes thatproduce angiostatin in vivo or in vitro.

[0042] It is a further object of the present invention to provideangiostatin or anti-angiostatin antibodies by direct injection ofangiostatin DNA into a human or animal needing such angiostatin oranti-angiostatin antibodies.

[0043] It is an object of present invention to provide a method fordetecting and quantifying the presence of an antibody specific for anangiostatin in a body fluid.

[0044] Still another object of the present invention is to provide acomposition consisting of antibodies to angiostatin that are selectivefor specific regions of the angiostatin molecule that do not recognizeplasminogen.

[0045] It is another object of the present invention to provide a methodfor the detection or prognosis of cancer.

[0046] It is another object of the present invention to provide acomposition for use in visualizing and quantitating sites of angiostatinbinding in vivo and in vitro.

[0047] It is yet another object of the present invention to provide acomposition for use in detection and quantification of angiostatinbiosynthesis.

[0048] It is yet another object of the present invention to provide atherapy for cancer that has minimal side effects.

[0049] Still another object of the present invention is to provide acomposition comprising angiostatin or an angiostatin protein linked to acytotoxic agent for treating or repressing the growth of a cancer.

[0050] Another object of the present invention is to provide a methodfor targeted delivery of angiostatin-related compositions to specificlocations.

[0051] Yet another object of the invention is to provide compositionsand methods useful for gene therapy for the modulation of angiogenicprocesses.

[0052] These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

[0053]FIG. 1 shows SEQ ID NO:1, the amino acid sequence of the wholemurine plasminogen.

[0054]FIG. 2 shows the beginning sequence of the angiostatin for murine(SEQ ID NO:2) and compares the murine sequence with corresponding human(SEQ ID NO:3), Rhesus monkey (SEQ ID NO:4), porcine (SEQ ID NO:5) andbovine (SEQ ID NO:6) plasminogen protein fragments. The mouse sequenceis listed first, followed by human, Rhesus, porcine and bovine.

[0055]FIG. 3 shows BrdU labeling index of tumor cells in the lung in thepresence or absence of a primary tumor.

[0056]FIG. 4 shows Matrigel analysis of the influence of a Lewis lungprimary tumor on bFGF driven angiogenesis in vivo.

[0057]FIG. 5 shows dose response curve for serum derived from micebearing Lewis lung carcinoma (LLC-Low) versus serum from normal mice.Bovine capillary endothelial cells were assayed in a bFGF-driven 72-hourproliferation assay.

[0058]FIG. 6 shows that both low and high metastatic tumors containendothelial mitogenic activity in their ascites, but only the lowmetastatic tumor line has endothelial inhibitory activity in the serum.

[0059]FIG. 7 shows a C4 Reverse Phase Chromatographic profile ofpartially purified serum or urine from tumor-bearing animals.

[0060]FIG. 8 shows surface lung metastases after the 13 day treatment ofmice with intact plasminogen molecule, active fraction from a lysinebinding site I preparation of human plasminogen, concentrated urine fromtumor bearing mice and concentrated urine from normal mice.

[0061]FIG. 9 shows lung weight after the 13 day treatment of mice withintact plasminogen molecule of human plasminogen, active fraction fromlysine binding site I preparation, concentrated urine from tumor bearingmice and concentrated urine from normal mice.

[0062]FIG. 10 is a schematic representation of the pTrcHis vector.

[0063]FIG. 11 depicts an immunoblot of E.coli expressed humanangiostatin from a 10 L scaled-up fermentation, probed with monoclonalantibody against human plasminogen kringle region 1-3. Arrow showsrecombinant human angiostatin. A) shows recombinant angiostatin elutedwith 0.2 M amino caproic acid; B) shows the last wash with 1× PBS of thelysine column; and C) shows clarified lysate from cracked cells.

[0064]FIG. 12. Is a graph depicting percent inhibition of growing bovinecapillary endothelial cells as a function of dilution of stock; A1, A2,B1, B2, and E are recombinant clones that express human angiostatinanit-angiogenesis activity; C1, C2, D1 and D2 controls are negativecontrols clones containing vector only without the human DNA sequencecoding for angiostatin.

[0065]FIG. 13 shows the inhibitory effect on proliferation ofrecombinant human angiostatin on bovine capillary endothelial cells invitro.

[0066]FIG. 14 shows the growth proliferation index and apoptotic indexafter removal of the primary tumor and treatment with saline or afumagillin analogue with anti-angiogenic activity

[0067]FIG. 15 shows the inhibition of growth of a T241 primary tumor inmice by treatment with human angiostatin in vivo with a single injectionof 40 mg/kg/day.

[0068]FIG. 16 shows the inhibition of growth of a LLC-LM primary tumorin mice by treatment with human angiostatin in vivo at two doses of 40mg/kg per dose (80 mg/kg/day).

[0069]FIG. 17 shows the effect of the removal of a Lewis lung carcinomaprimary tumor on the growth of its lung metastases.

[0070]FIG. 18 shows the growth proliferation and apoptotic index aftertumor resection

[0071]FIG. 19 shows the effect of administration of angiostatin proteinto mice having implated T241 fibrosarcoma cells on total tumor volume asa function of time.

[0072]FIG. 20 shows the effect of administration of angiostatin proteinto mice having implated Lewis lung carcinoma (LM) cells on total tumorvolume as a function of time.

[0073]FIG. 21 shows the effect of administration of angiostatin proteinto mice having implated reticulum cell sarcoma cells on total tumorvolume as a function of time.

[0074]FIG. 22 shows the effect of administration of angiostatin proteinto immunodeficient SCID mice having implated human prostate carcinomaPC-3 cells on total tumor volume as a function of time over a 24 dayperiod.

[0075]FIG. 23 shows the effect of administration of angiostatin proteinto immunodeficient SCID mice having implated human breast carcinomaMDA-MB cells on total tumor volume as a function of time over a 24 dayperiod.

[0076]FIG. 24 is a schematic representation of cloning of the mouse DNAsequence coding for mouse angiostatin protein derived from mouseplasminogen cDNA. The mouse angiostatin encompasses mouse plasminogenkringle regions 1-4. PCR means polymerase chain reaction; P1 is the5′-end oligonucleotide primre for PCR; P2 is the 3′-end oligonucleotideprimre for PCR; SS designates the signal sequence; ATG is thetranslation initiation codon; TAA is the translation stop codon; HArepresents the hemagglutinin epitope tag (YPYDVPDYASL); K1, K2, K3 andK4 represent mouse plasminogen kringle regions 1, 2, 3 and 4respectively. CMV is the cytomegalovirus promoter; T7 is the bacteriaphage promoter; PA represents pre-activation proteins; and SP6 is the Sp6 promoter.

[0077]FIG. 25 depicts the number of cells as a function of days fornon-transfected cells (mock); cells transfected with the vector alone,without the DNA sequence coding for angiostatin (Vector 5), and twoangiostatin expressing clones (AST 31 and AST 37). Panel (a) representsthe results of transfection of T241 cells. Panel (b) represents theresults of LL2 cells.

[0078]FIG. 26 shows the results of culture medium derived from E. colicells containing the angiostatin clone on cell number. Non-transfectedcells (mock); cells transfected with the vector alone, without the DNAsequence coding for angiostatin (Vector 5), and three angiostatinexpressing clones (AST 25, AST 31 and AST 37). Panel (a) represents theresults of incubation of culture medium from control (mock) and allangiostatin clones (expressing and non-expressing) on cell number. Panel(b) represents the results of incubation of culture medium from control(mock), vector alone (vector 6) and angiostatin clones expressing mouseangiostatin on cell number. Panel (c) represents the results ofincubation of purified culture medium from control (mock) andangiostatin clones expressing mouse angiostatin on cell number, whereinthe culture medium was purified over a lysine-sepharose colume to yieldlysine binding components.

[0079]FIG. 27 shows the effect on total tumor volume as a function oftime of implanting T241 fibrosarcoma cells in mice, where thefibrosarcoma cells have been transfected with a vector containing a DNAsequence coding for angiostatin protein, and where the vector is capableof expressing angiostatin protein. “Non-transfected” representsunaltered T241 fibrosarcoma cells implanted in mice. “Vector 6”represents T241 fibrosarcoma cells transfected with the vector only,which does not contain the DNA sequence coding for angiostatin protein,implanted in mice. “Clone 25, Clone 31 and Clone 37” represent threeangiostatin-producing clones of T241 fibrosarcoma cells transfected witha vector containg the DNA sequence coding for angiostation proteinimplanted in mice.

[0080]FIG. 28 shows a schematic representation of the structure of humanplasminogen and its kringle fragments. Human plaminogen is a singlechain protein containing 791 amino acids with one side of N-linkedglycosylation at Asn²⁸⁹. The non-protease region of human plasminogenconsisting of the N-terminal 561 amino acids existing in five separatedomains, termed kringles as shown in circles (K1, K2, K3, K4 and K5),along with proteins that separate these structures. Each tripledisulfide bonded kringle contains 80 amino acids. Angiostatin covers thefirst 4 of these kringle domains (K1-4), kringle 3 (K1-3) and kringle 4(K4) are obtained by digestion of human plasminogen with elastase. Therest of the kringle fragments are recombinant proteins expressed in E.coli. SS=signal sequence. PA=preactivation protein.

[0081]FIG. 29 shows a SDS-PAGE analysis of purified recombinant andnative kringle fragments of plasminogen under reducing conditions. (A)Individual recombinant kringle fragments purified from E. coli bacteriallysates were loaded onto a 15% SDS gel followed by staining withCoomassie blue. Approximately 5 μg of each protein was loaded per lane.(lane 2=kringle 1 (K1); lane 3=kringle 2 (K2); lane 4=kringle 3 (K3);lane 5=kringle 4 (K4); lane 1=molecular weight markers). (B) Purifiedlarge kringle fragments were stained with Coomassie blue. Kringles 1-4(lane 2) and kringles 1-3 (lane 3) were obtained by digestion of humanplasminogen with elastase and purified by lysine-Sepharosechromatography. Recombinant fragment of kringles 2-3 (lane 4) wasexpressed in E. coli and re-folded in vitro. Molecular weight markersare indicated on the left (lane 1).

[0082]FIG. 30 shows an inhibition of endothelial cell proliferation byrecombinant individual kringle fragments of angiostatin. Kringlefragments were assayed on bovine capillary endothelial cells in thepresence of 1 ng/ml bFGF for 72 hours. (A) Anti-endothelial cellproliferative effects of two lysine-binding kringles, rK1 and rK4. Thehigh-affinity lysine binding kringle, K1 (-o-), inhibited BCE cellproliferation in a dose-dependent manner. The intermediate-affinitylysine binding kringle, K4 (--), showed only little inhibitory effectat high concentrations. (B) Inhibition of BCE cell proliferation bynon-lysine binding K2 and K3. Both K2 (-▪-) and K3 (-□-) inhibited BCEcell proliferation in a dose-dependent manner. Data represents themean+/−SEM of triplicates.

[0083]FIG. 31 shows an anti-endothelial proliferation activity of largekringle fragments of angiostatin. Proteolytic fragments, K1-4(angiostatin) (-o-) and K1-3 (-▪-), inhibited BCE cell proliferation ina dose-dependent manner. Recombinant K2-3 (--) fragments exhibited aless potent inhibition than those of K1-3 and K1-4. Data represents themean of three determinations (+/−SEM) as percentages of inhibition.

[0084]FIG. 32 shows an additive inhibitory activity of recombinantkringle 2 and kringle 3. (A) The intact fragment of rK2-3 (also see FIG.31) displayed a weak inhibitory effect only at the concentration of 320nM. At the same concentration, an additive inhibition was seen whenmutant fragments of rK2 cysteine replaced by serine at the position of169) and K3 (cysteine replaced by serine at the position of 297) wereassayed together on BCE cells. Each value represents the mean+/−SEM oftriplicates. (B) Schematic structure and amino acid sequence of K2 andK3. An inter-chain kringle disulfide bond was previously reported to bepresent between cysteine¹⁶⁹ of K2 and cysteine²⁹⁷ of K3 (Söhndel, S.,Hu, C.-K., Marti, D., Affolter, M., Schaller, J., Llinas, M., andRickli, E. E. (1996) Biochem. in press).

[0085]FIG. 33 shows an inhibition of endothelial proliferation bycombinatorial kringle fragments. The assay was performed with aconcentration of 320 nM for each kringle fragment. Values represent themean of three determinations (+/−SEM) as percentages of inhibition. (A)Inhibitory effects of fragments by combination of various individualkringles. (B) Combinatorial inhibitory activity of combined kringlefragments.

[0086]FIG. 34 shows an inhibitory activity of angiostatin on endothelialcells after reduction and alkylation. (A) SDS-PAGE analysis of thereduced (lane 2) and non-reduced (lane 1) forms of human angiostatin.Purified human angiostatin was reduced with DTT followed by alkylationof the protein with an excess amount of iodoacetamide. The treatedsamples were dialyzed and assayed on BCE cells. (B) Inhibition of BCEcell proliferation by reduced and non-reduced forms of angiostatin at aconcentration of 320 nM. Data represents the mean of inhibition+/−SEM oftriplicates.

[0087]FIG. 35 shows an amino acid sequence alignment of putative kringledomains of human angiostatin. The sequences of four kringle domains werealigned according to their conserved cysteines. Identical and conservedamino acids are shaded. The boxed amino acids in kringle 4 show thepositively charged double lysines adjacent to conserved cysteineresidues of 22 and 80.

[0088]FIG. 36 shows lysine-binding characteristics and reactivity ofexpressed angiostatin.

[0089]FIG. 36A shows a Coomassie stained gel (40 μl load).

[0090]FIG. 36B shows an immunoblot (20 μl load) of similar gel. Lane:1shows broth from shake flasks of induced cultures showing angiostatinprotein at about 50 kD and a few other proteins. Broth from inducedcultures is diluted 1:1 with buffer and directly loaded ontolysine-sepharose. Lane:2 shows the unbound fraction that passed throughthe lysine column. All angiostatin protein expressed by P. pastorisbinds to the lysine column. Lane:3 shows specific elution with 0.2 Mamino caproic acid showing that P. pastoris expressed angiostatinprotein binds lysine and can be purified in a single step to homogeneityover a lysine-sepharose. Also, the P. pastoris expressed angiostatinprotein is recognized by a conformationally dependent monoclonalantibody (VAP) raised against kringles 1 to 3.

[0091]FIG. 37 shows P. pastoris expressed angiostatin protein is seen asa doublet that migrates at 49 kD and 51.5 kD on denatured unreducedSDS-PAGE Coomassie stained gels. Removing the single N-linked complexchain from the expressed angiostatin protein with N-glycanase specificfor high mannose structures results in a single band of 49.5 kD. Panel Aand panel B show a Coomassie stained gel and an immunoblot of a similargel respectively. Lane:1 shows a purified P. pastoris expressedangiostatin protein. Lane:2 shows a purified P. pastoris expressedangiostatin protein incubated in digestion conditions withoutN-glycanase. Lane:3 shows purified P. pastoris expressed angiostatinprotein digested with N-glycanase.

[0092]FIG. 38A shows 4 μg of purified P. pastoris expressed angiostatinprotein as a doublet on a Coomassie gel.

[0093]FIG. 38B shows that the purified recombinant inhibits BCEproliferation. The BCE assay cell counts obtained after 72 hours isshown, in the presence () or absence (o) of bFGF, and in the presenceof bFGF with PBS as control (Δ), and in the presence of bFGF with P.pastoris expressed angiostatin protein (Δ).

[0094]FIG. 38C shows that the inhibition is dose dependent.

[0095]FIG. 39 shows P. pastoris expressed purified angiostatin was givensystemically (subcutaneous) to mice with primary tumors.

[0096]FIGS. 39A and B show the number of metastases and the lung weightsrespectively of mice treated daily with saline or P. pastoris expressedangiostatin or plasminogen derived angiostatin protein. In contrast tothe lungs of mice treated with saline, lungs of mice treated with P.pastoris expressed angiostatin protein or with plasminogen derivedangiostatin protein were non-vascularized and metastases were potentlysuppressed.

[0097]FIG. 40 shows that the lungs of mice treated with P. pastorisexpressed angiostatin were pink with micrometastases while the lungs ofthe saline control group were completely covered with vascularizedmetastases.

DETAILED DESCRIPTION

[0098] The present invention includes compositions and methods for thedetection and treatment of diseases and processes that are mediated byor associated with angiogenesis. The composition is angiostatin, whichcan be isolated from body fluids including, but not limited to, serum,urine and ascites, or synthesized by chemical or biological methods(e.g. cell culture, recombinant gene expression, protein synthesis, andin vitro enzymatic catalysis of plasminogen or plasmin to yield activeangiostatin). Recombinant techniques include gene amplification from DNAsources using the polymerase chain reaction (PCR), and geneamplification from RNA sources using reverse transcriptase/PCR.Angiostatin inhibits the growth of blood vessels into tissues such asunvascularized or vascularized tumors.

[0099] The present invention also encompasses a composition comprising,a vector containing a DNA sequence encoding angiostatin, wherein thevector is capable of expressing angiostatin when present in a cell, acomposition comprising a cell containing a vector, wherein the vectorcontains a DNA sequence encoding angiostatin or fragments or analogsthereof, and wherein the vector is capable of expressing angiostatinwhen present in the cell, and a method comprising, implanting into ahuman or non-human animal a cell containing a vector, wherein the vectorcontains a DNA sequence encoding angiostatin, and wherein the vector iscapable of expressing angiostatin when present in the cell.

[0100] Still further, the present invention encompasses angiostatin,angiostatin fragments, angiostatin antisera, angiostatin receptoragonists or angiostatin receptor antagonists that are combined withpharmaceutically acceptable excipients, and optionally sustained-releasecompounds or compositions, such as biodegradable polymers, to formtherapeutic compositions. In particular, the invention includes acomposition comprising an antibody that specifically binds toangiostatin, wherein the antibody does not bind to plasminogen.

[0101] More particularly, the present invention includes a proteindesignated angiostatin that has a molecular weight of approximately 38to 45 kilodaltons (kD) that is capable of overcoming the angiogenicactivity of endogenous growth factors such as bFGF, in vitro.Angiostatin is a protein having a molecular weight of betweenapproximately 38 kilodaltons and 45 kilodaltons as determined byreducing polyacrylamide gel electrophoresis and having an amino acidsequence substantially similar to that of a murine plasminogen fragmentbeginning at amino acid number 98 of an intact murine plasminogenmolecule. Numbering of amino acids herein corresponds to theconventioanl system of numbering from the beginning methionine of theplasminogen molecule.

[0102] The term “substantially similar,” when used in reference toangiostatin amino acid sequences, means an amino acid sequence havinganti-angiogenic activity and having a molecular weight of approximately38 kD to 45 kD, which also has a high degree of sequence homology to theprotein fragment of mouse plasminogen beginning approximately at aminoacid number 98 in mouse plasminogen and weighing 38 kD to 45 kD. A highdegree of homology means at least approximately 60% amino acid homology,desirably at least approximately 70% amino acid homology, and moredesirably at least approximately 80% amino acid homology. The term“endothelial inhibiting activity” as used herein means the capability ofa molecule to inhibit angiogenesis in general and, for example, toinhibit the growth of bovine capillary endothelial cells in culture inthe presence of fibroblast growth factor.

[0103] The amino acid sequence of the complete murine plasminogenmolecule is shown in FIG. 1 and in SEQ ID NO:1. The sequence forangiostatin protein can begin approximately at amino acid 98. Activehuman angiostatin, howvere, can also begin at a variety of alternativepositions. The examples demonstrate that genetic constructs encodingactive angiostatin protein can begin at amino acid 93 or 102, forexample.

[0104] The amino acid sequence of the first 339 amino acids of anangiostatin from mouse is shown in FIG. 2, (SEQ ID NO:2), and iscompared with the sequences of corresponding plasminogen proteinfragments from human (SEQ ID NO:3, Rhesus monkey (SEQ ID NO:4), porcine(SEQ ID NO:5) and bovine (SEQ ID NO:6) plasminogen. Given that thesesequences are identical in well over 50% of their amino acids, it is tobe understood that the amino acid sequence of the angiostatin issubstantially similar among species. The total number of amino acids inangiostatin is not known precisely but is defined by the molecularweight of the active molecule. The amino acid sequence of theangiostatin of the present invention may vary depending upon from whichspecies the plasminogen molecule is derived. Thus, although theangiostatin of the present invention that is derived from humanplasminogen has a slightly different sequence than angiostatin derivedfrom mouse, it has anti-angiogenic activity as shown in a mouse tumormodel.

[0105] Angiostatin has been shown to be capable of inhibiting the growthof endothelial cells in vitro. Angiostatin does not inhibit the growthof cell lines derived from other cell types. Specifically, angiostatinhas no effect on Lewis lung carcinoma cell lines, mink lung epithelium,3T3 fibroblasts, bovine aortic smooth muscle cells, bovine retinalpigment epithelium, MDCk cells (canine renal epithelium), WI38 cells(human fetal lung fibroblasts) EFN cells (murine fetal fibroblasts) andLM cells (murine connective tissue). Endogenous angiostatin in a tumorbering mouse is effective at inhibiting metastases at a systemicconcentration of approximately 10 mg angiostatin/kg body weight.

[0106] Angiostatin has a specific three dimensional conformation that isdefined by the kringle regions of the plasminogen molecule. (Robbins, K.C., “The plasminogen-plasmin enzyme system” Hemostasis and Thrombosis,Basic Principles and Practice, 2nd Edition, ed. by Colman, R. W. et al.J. B. Lippincott Company, pp. 340-357, 1987) There are five such kringleregions, which are conformationally related motifs and have substantialsequence homology, in the NH₂ terminal portion of the plasminogenmolecule. The three dimensional conformation of functional angiostatinis believed to encompass plasminogen kringle regions 1 through 5. Eachkringle region of the plasminogen molecule contains approximately 80amino acids and contains 3 disulfide bonds. This cysteine motif is knownto exist in other biologically active proteins. These proteins include,but are not limited to, prothrombin, hepatocyte growth factor, scatterfactor and macrophage stimulating protein. (Yoshimura, T, et al.,“Cloning, sequencing, and expression of human macrophage stimulatingprotein (MSP, MST1) confirms MSP as a member of the family of kringleproteins and locates the MSP gene on Chromosome 3” J. Biol. Chem., Vol.268, No. 21, pp. 15461-15468, 1993). It is contemplated that anyisolated protein or protein having a three dimensional kringle-likeconformation or cysteine motif that has anti-angiogenic activity invivo, is part of the present invention.

[0107] The present invention also includes the detection of theangiostatin in body fluids and tissues for the purpose of diagnosis orprognosis of diseases such as cancer. The present invention alsoincludes the detection of angiostatin binding sites and receptors incells and tissues. The present invention also includes methods oftreating or preventing angiogenic diseases and processes including, butnot limited to, arthritis and tumors by stimulating the production ofangiostatin, and/or by administering substantially purified angiostatin,or angiostatin agonists or antagonists, and/or angiostatin antisera orantisera directed against angiostatin antisera to a patient. Additionaltreatment methods include administration of angiostatin, angiostatinfragments, angiostatin analogs, angiostatin antisera, or angiostatinreceptor agonists and antagonists linked to cytotoxic agents. It is tobe understood that the angiostatin can be animal or human in origin.Angiostatin can also be produced synthetically by chemical reaction orby recombinant techniques in conjunction with expression systems.Angiostatin can also be produced by enzymatically cleaving isolatedplasminogen or plasmin to generate proteins having anti-angiogenicactivity. Angiostatin may also be produced by compounds that mimic theaction of endogenous enzymes that cleave plasminogen to angiostatin.Angiostatin production may also be modulated by compounds that affectthe activity of plasminogen cleaving enxymes.

[0108] Passive antibody therapy using antibodies that specifically bindangiostatin can be employed to modulate angiogenic-dependent processessuch as reproduction, development, and wound healing and tissue repair.In addition, antisera directed to the Fab regions of angiostatinantibodies can be administered to block the ability of endogenousangiostatin antisera to bind angiostatin.

[0109] The present invention also encompasses gene therapy whereby thegene encoding angiostatin is regulated in a patient. Various methods oftransferring or delivering DNA to cells for expression of the geneproduct protein, otherwise referred to as gene therapy, are disclosed inGene Transfer into Mammalian Somatic Cells in vivo, N. Yang, Crit. Rev.Biotechn. 12(4): 335-356 (1992), which is hereby incorporated byreference. Gene therapy encompasses incorporation of DNA sequences intosomatic cells or germ line cells for use in either ex vivo or in vivotherapy. Gene therapy functions to replace genes, augment normal orabnormal gene function, and to combat infectious diseases and otherpathologies.

[0110] Strategies for treating these medical problems with gene therapyinclude therapeutic strategies such as identifying the defective geneand then adding a functional gene to either replace the function of thedefective gene or to augment a slightly functional gene; or prophylacticstrategies, such as adding a gene for the product protein that willtreat the condition or that will make the tissue or organ moresusceptible to a treatment regimen. As an example of a prophylacticstrategy, a gene such as angiostatin may be placed in a patient and thusprevent occurrence of angiogenesis; or a gene that makes tumor cellsmore susceptible to radiation could be inserted and then radiation ofthe tumor would cause increased killing of the tumor cells.

[0111] Many protocols for transfer of angiostatin DNA or angiostatinregulatory sequences are envisioned in this invention. Transfection ofpromoter sequences, other than one normally found specificallyassociated with angiostatin, or other sequences which would increaseproduction of angiostatin protein are also envisioned as methods of genetherapy. An example of this technology is found in TranskaryoticTherapies, Inc., of Cambridge, Mass., using homologous recombination toinsert a “genetic switch” that turns on an erythropoietin gene in cells.See Genetic Engineering News, Apr. 15, 1994. Such “genetic switches”could be used to activate angiostatin (or the angiostatin receptor) incells not normally expressing angiostatin (or the angiostatin receptor).

[0112] Gene transfer methods for gene therapy fall into three broadcategories-physical (e.g., electroporation, direct gene transfer andparticle bombardment), chemical (lipid-based carriers, or othernon-viral vectors) and biological (virus-derived vector and receptoruptake). For example, non-viral vectors may be used which includeliposomes coated with DNA. Such liposome/DNA complexes may be directlyinjected intravenously into the patient. It is believed that theliposome/DNA complexes are concentrated in the liver where they deliverthe DNA to macrophages and Kupffer cells. These cells are long lived andthus provide long term expression of the delivered DNA. Additionally,vectors or the “naked” DNA of the gene may be directly injected into thedesired organ, tissue or tumor for targeted delivery of the therapeuticDNA.

[0113] Gene therapy methodologies can also be described by deliverysite. Fundamental ways to deliver genes include ex vivo gene transfer,in vivo gene transfer, and in vitro gene transfer. In ex vivo genetransfer, cells are taken from the patient and grown in cell culture.The DNA is transfected into the cells, the transfected cells areexpanded in number and then reimplanted in the patient. In in vitro genetransfer, the transformed cells are cells growing in culture, such astissue culture cells, and not particular cells from a particularpatient. These “laboratory cells” are transfected, the transfected cellsare selected and expanded for either implantation into a patient or forother uses.

[0114] In vivo gene transfer involves introducing the DNA into the cellsof the patient when the cells are within the patient. Methods includeusing virally mediated gene transfer using a noninfectious virus todeliver the gene in the patient or injecting naked DNA into a site inthe patient and the DNA is taken up by a percentage of cells in whichthe gene product protein is expressed. Additionally, the other methodsdescribed herein, such as use of a “gene gun,” may be used for in vitroinsertion of angiostatin DNA or angiostatin regulatory sequences.

[0115] Chemical methods of gene therapy may involve a lipid basedcompound, not necessarily a liposome, to ferry the DNA across the cellmembrane. Lipofectins or cytofectins, lipid-based positive ions thatbind to negatively charged DNA, make a complex that can cross the cellmembrane and provide the DNA into the interior of the cell. Anotherchemical method uses receptor-based endocytosis, which involves bindinga specific ligand to a cell surface receptor and enveloping andtransporting it across the cell membrane. The ligand binds to the DNAand the whole complex is transported into the cell. The ligand genecomplex is injected into the blood stream and then target cells thathave the receptor will specifically bind the ligand and transport theligand-DNA complex into the cell.

[0116] Many gene therapy methodologies employ viral vectors to insertgenes into cells. For example, altered retrovirus vectors have been usedin ex vivo methods to introduce genes into peripheral andtumor-infiltrating lymphocytes, hepatocytes, epidermal cells, myocytes,or other somatic cells. These altered cells are then introduced into thepatient to provide the gene product from the inserted DNA.

[0117] Viral vectors have also been used to insert genes into cellsusing in vivo protocols. To direct tissue-specific expression of foreigngenes, cis-acting regulatory elements or promoters that are known to betissue specific can be used. Alternatively, this can be achieved usingin situ delivery of DNA or viral vectors to specific anatomical sites invivo. For example, gene transfer to blood vessels in vivo was achievedby implanting in vitro transduced endothelial cells in chosen sites onarterial walls. The virus infected surrounding cells which alsoexpressed the gene product. A viral vector can be delivered directly tothe in vivo site, by a catheter for example, thus allowing only certainareas to be infected by the virus, and providing long-term, sitespecific gene expression. In vivo gene transfer using retrovirus vectorshas also been demonstrated in mammary tissue and hepatic tissue byinjection of the altered virus into blood vessels leading to the organs.

[0118] Viral vectors that have been used for gene therapy protocolsinclude but are not limited to, retroviruses, other RNA viruses such aspoliovirus or Sindbis virus, adenovirus, adeno-associated virus, herpesviruses, SV 40, vaccinia and other DNA viruses. Replication-defectivemurine retroviral vectors are the most widely utilized gene transfervectors. Murine leukemia retroviruses are composed of a single strandRNA complexed with a nuclear core protein and polymerase (pol) enzymes,encased by a protein core (gag) and surrounded by a glycoproteinenvelope (env) that determines host range. The genomic structure ofretroviruses include the gag, pol, and env genes enclosed at by the 5′and 3′ long terminal repeats (LTR). Retroviral vector systems exploitthe fact that a minimal vector containing the 5′ and 3′ LTRs and thepackaging signal are sufficient to allow vector packaging, infection andintegration into target cells providing that the viral structuralproteins are supplied in trans in the packaging cell line. Fundamentaladvantages of retroviral vectors for gene transfer include efficientinfection and gene expression in most cell types, precise single copyvector integration into target cell chromosomal DNA, and ease ofmanipulation of the retroviral genome.

[0119] The adenovirus is composed of linear, double stranded DNAcomplexed with core proteins and surrounded with capsid proteins.Advances in molecular virology have led to the ability to exploit thebiology of these organisms to create vectors capable of transducingnovel genetic sequences into target cells in vivo. Adenoviral-basedvectors will express gene product proteins at high levels. Adenoviralvectors have high efficiencies of infectivity, even with low titers ofvirus. Additionally, the virus is fully infective as a cell free virionso injection of producer cell lines are not necessary. Another potentialadvantage to adenoviral vectors is the ability to achieve long termexpression of heterologous genes in vivo.

[0120] Mechanical methods of DNA delivery include fusogenic lipidvesicles such as liposomes or other vesicles for membrane fusion, lipidparticles of DNA incorporating cationic lipid such as lipofectin,polylysine-mediated transfer of DNA, direct injection of DNA, such asmicroinjection of DNA into germ or somatic cells, pneumaticallydelivered DNA-coated particles, such as the gold particles used in a“gene gun,” and inorganic chemical approaches such as calcium phosphatetransfection. Another method, ligand-mediated gene therapy, involvescomplexing the DNA with specific ligands to form ligand-DNA conjugates,to direct the DNA to a specific cell or tissue.

[0121] It has been found that injecting plasmid DNA into muscle cellsyields high percentage of the cells which are transfected and havesustained expression of marker genes. The DNA of the plasmid may or maynot integrate into the genome of the cells. Non-integration of thetransfected DNA would allow the transfection and expression of geneproduct proteins in terminally differentiated, non-proliferative tissuesfor a prolonged period of time without fear of mutational insertions,deletions, or alterations in the cellular or mitochondrial genome.Long-term, but not necessarily permanent, transfer of therapeutic genesinto specific cells may provide treatments for genetic diseases or forprophylactic use. The DNA could be reinjected periodically to maintainthe gene product level without mutations occurring in the genomes of therecipient cells. Non-integration of exogenous DNAs may allow for thepresence of several different exogenous DNA constructs within one cellwith all of the constructs expressing various gene products.

[0122] Particle-mediated gene transfer methods were first used intransforming plant tissue. With a particle bombardment device, or “genegun,” a motive force is generated to accelerate DNA-coated high densityparticles (such as gold or tungsten) to a high velocity that allowspenetration of the target organs, tissues or cells. Particle bombardmentcan be used in in vitro systems, or with ex vivo or in vivo techniquesto introduce DNA into cells, tissues or organs.

[0123] Electroporation for gene transfer uses an electrical current tomake cells or tissues susceptible to electroporation-mediated genetransfer. A brief electric impulse with a given field strength is usedto increase the permeability of a membrane in such a way that DNAmolecules can penetrate into the cells. This technique can be used in invitro systems, or with ex vivo or in vivo techniques to introduce DNAinto cells, tissues or organs.

[0124] Carrier mediated gene transfer in vivo can be used to transfectforeign DNA into cells. The carrier-DNA complex can be convenientlyintroduced into body fluids or the bloodstream and then sitespecifically directed to the target organ or tissue in the body. Bothliposomes and polycations, such as polylysine, lipofectins orcytofectins, can be used. Liposomes can be developed which are cellspecific or organ specific and thus the foreign DNA carried by theliposome will be taken up by target cells. Injection of immunoliposomesthat are targeted to a specific receptor on certain cells can be used asa convenient method of inserting the DNA into the cells bearing thereceptor. Another carrier system that has been used is theasialoglycoportein/polylysine conjugate system for carrying DNA tohepatocytes for in vivo gene transfer.

[0125] The transfected DNA may also be complexed with other kinds ofcarriers so that the DNA is carried to the recipient cell and thenresides in the cytoplasm or in the nucleoplasm. DNA can be coupled tocarrier nuclear proteins in specifically engineered vesicle complexesand carried directly into the nucleus.

[0126] Gene regulation of angiostatin may be accomplished byadministering compounds that bind to the angiostatin gene, or controlregions associated with the angiostatin gene, or its corresponding RNAtranscript to modify the rate of transcription or translation.Additionally, cells transfected with a DNA sequence encoding angiostatinmay be administered to a patient to provide an in vivo source ofangiostatin. For example, cells may be transfected with a vectorcontaining a nucleic acid sequence encoding angiostatin.

[0127] The term “vector” as used herein means a carrier that can containor associate with specific nucleic acid sequences, which functions totransport the specific nucleic acid sequences into a cell. Examples ofvectors include plasmids and infective microorganisms such as viruses,or non-viral vectors such as ligand-DNA conjugates, liposomes, lipid-DNAcomplexes. It may be desirable that a recombinant DNA moleculecomprising an angiostatin DNA sequence is operatively linked to anexpression control sequence to form an expression vector capable ofexpressing angiostatin. The transfected cells may be cells derived fromthe patient's normal tissue, the patient's diseased tissue, or may benon-patient cells.

[0128] For example, tumor cells removed from a patient can betransfected with a vector capable of expressing the angiostatin proteinof the present invention, and re-introduced into the patient. Thetransfected tumor cells produce angiostatin levels in the patient thatinhibit the growth of the tumor. Patients may be human or non-humananimals. Cells may also be transfected by non-vector, or physical orchemical methods known in the art such as electroporation, ionoporation,or via a “gene gun.” Additionally, angiostatin DNA may be directlyinjected, without the aid of a carrier, into a patient. In particular,angiostatin DNA may be injected into skin, muscle or blood.

[0129] The gene therapy protocol for transfecting angiostatin into apatient may either be through integration of the angiostatin DNA intothe genome of the cells, into minichromosomes or as a separatereplicating or non-replicating DNA construct in the cytoplasm ornucleoplasm of the cell. Angiostatin expression may continue for along-period of time or may be reinjected periodically to maintain adesired level of the angiostatin protein in the cell, the tissue ororgan or a determined blood level.

[0130] Angiostatin can be isolated on an HPLC C4 column (see Table 3).The angiostatin protein is eluted at 30 to 35% in an acetonitrilegradient. On a sodium dodecyl sulfate polyacrylamide gel electrophoresis(PAGE) gel under reducing conditions, the protein band with activityeluted as a single peak at approximately 38 kilodaltons.

[0131] The inventors have shown that a growing primary tumor isassociated with the release into the blood stream of specificinhibitor(s) of endothelial cell proliferation, including angiostatinwhich can suppress angiogenesis within a metastasis and thereby inhibitthe growth of the metastasis itself. The source of the angiostatinassociated with the primary tumor is not known. The compound may beproduced by degradation of plasminogen by a specific protease, orangiostatin could be produced by expression of a specific gene codingfor angiostatin.

[0132] The angiogenic phenotype of a primary tumor depends on productionof angiogenic proteins in excess of endothelial cell inhibitors whichare elaborated by normal cells, but are believed to be down-regulatedduring transformation to neoplasia. While production of angiostatin maybe down-regulated in an individual tumor cell relative to production byits parent cell type, the total amount of inhibitor elaborated by thewhole tumor may be sufficient to enter the circulation and suppressendothelial growth at remote sites of micrometastases. Angiostatinremains in the circulation for a significantly longer time than theangiogenic protein(s) released by a primary tumor. Thus, the angiogenicproteins appear to act locally, whereas angiostatin acts globally andcirculates in the blood with a relatively long half-life. The half-lifeof the angiostatin is approximately 12 hours to 5 days.

[0133] Although not wanting to be bound by the following hypothesis, itis believed that when a tumor becomes angiogenic it releases one or moreangiogenic proteins (e.g., aFGF, bFGF, VEGF, IL-8, GM-CSF, etc.), whichact locally, target endothelium in the neighborhood of a primary tumorfrom an extravascular direction, and do not circulate (or circulate witha short half-life). These angiogenic proteins must be produced in anamount sufficient to overcome the action of endothelial cell inhibitor(inhibitors of angiogenesis) for a primary tumor to continue to expandits population. Once such a primary tumor is growing well, it continuesto release endothelial cell inhibitors into the circulation. Accordingto this hypothesis, these inhibitors act remotely at a distance from theprimary tumor, target capillary endothelium of a metastasis from anintravascular direction, and continue to circulate. Thus, just at thetime when a remote metastasis might begin to initiate angiogenesis, thecapillary endothelium in its neighborhood could be inhibited by incomingangiostatin.

[0134] Once a primary tumor has reached sufficient size to causeangiostatin to be released continuously into the circulation, it isdifficult for a second tumor implant (or a micrometastasis) to initiateor increase its own angiogenesis. If a second tumor implant (e.g., intothe subcutaneous space, or into the cornea, or intravenously to thelung) occurs shortly after the primary tumor is implanted, the primarytumor will not be able to suppress the secondary tumor (becauseangiogenesis in the secondary tumor will already be well underway). Iftwo tumors are implanted simultaneously (e.g., in opposite flanks), theinhibitors may have an equivalent inhibiting effect on each other.

[0135] The angiostatin of the present invention can be:

[0136] (i) Administered to tumor-bearing humans or animals asanti-angiogenic therapy;

[0137] (ii) Monitored in human or animal serum, urine, or tissues asprognostic markers; and

[0138] (iii) Used as the basis to analyze serum and urine of cancerpatients for similar angiostatic molecules.

[0139] It is contemplated as part of the present invention thatangiostatin can be isolated from a body fluid such as blood or urine ofpatients or the angiostatin can be produced by recombinant DNA methodsor synthetic protein chemical methods that are well known to those ofordinary skill in the art. Protein purification methods are well knownin the art and a specific example of a method for purifying angiostatin,and assaying for inhibitor activity is provided in the examples below.Isolation of human endogenous angiostatin is accomplished using similartechniques.

[0140] One example of a method of producing angiostatin usingrecombinant DNA techniques entails the steps of (1) identifying andpurifying angiostatin as discussed above, and as more fully describedbelow, (2) determining the N-terminal amino acid sequence of thepurified inhibitor, (3) synthetically generating 5′ and 3′ DNAoligonucleotide primers for the angiostatin sequence, (4) amplifying theangiostatin gene sequence using polymerase, (5) inserting the amplifiedsequence into an appropriate vector such as an expression vector, (6)inserting the gene containing vector into a microorganism or otherexpression system capable of expressing the inhibitor gene, and (7)isolating the recombinantly produced inhibitor. Appropriate vectorsinclude viral, bacterial and eukaryotic (such as yeast) expressionvectors. The above techniques are more fully described in laboratorymanuals such as “Molecular Cloning: A Laboratory Manual” Second Editionby Sambrook et al., Cold Spring Harbor Press, 1989. The DNA sequence ofhuman plasminogen has been published (Browne, M. J., et al., “Expressionof recombinant human plasminogen and aglycoplasminogen in HeLa cells”Fibrinolysis Vol.5 (4). 257-260, 1991) and is incorporated herein byreference

[0141] The gene for angiostatin may also be isolated from cells ortissue (such as tumor cells) that express high levels of angiostatin by(1) isolating messenger RNA from the tissue, (2) using reversetranscriptase to generate the corresponding DNA sequence and then (3)using the polymerase chain reaction (PCR) with the appropriate primersto amplify the DNA sequence coding for the active angiostatin amino acidsequence.

[0142] Yet another method of producing angiostatin, or biologicallyactive fragments thereof, is by protein synthesis. Once a biologicallyactive fragment of an angiostatin is found using the assay systemdescribed more fully below, it can be sequenced, for example byautomated protein sequencing methods. Alternatively, once the gene orDNA sequence which codes for angiostatin is isolated, for example by themethods described above, the DNA sequence can be determined using manualor automated sequencing methods well know in the art. The nucleic acidsequence in turn provides information regarding the amino acid sequence.Thus, if the biologically active fragment is generated by specificmethods, such as tryptic digests, or if the fragment is N-terminalsequenced, the remaining amino acid sequence can be determined from thecorresponding DNA sequence.

[0143] Once the amino acid sequence of the protein is known, thefragment can be synthesized by techniques well known in the art, asexemplified by “Solid Phase Protein Synthesis: A Practical Approach” E.Atherton and R. C. Sheppard, IRL Press, Oxford, England. Similarly,multiple fragments can be synthesized which are subsequently linkedtogether to form larger fragments. These synthetic protein fragments canalso be made with amino acid substitutions at specific locations to testfor agonistic and antagonistic activity in vitro and in vivo. Proteinfragments that possess high affinity binding to tissues can be used toisolate the angiostatin receptor on affinity columns. Isolation andpurification of the angiostatin receptor is a fundamental step towardselucidating the mechanism of action of angiostatin. Isolation of anangiostatin receptor and identification of angiostatin agonists andantagonists will facilitate development of drugs to modulate theactivity of the angiostatin receptor, the final pathway to biologicalactivity. Isolation of the receptor enables the construction ofnucleotide probes to monitor the location and synthesis of the receptor,using in situ and solution hybridization technology. Further, the genefor the angiostatin receptor can be isolated, incorporated into anexpression vector and transfected into cells, such as patient tumorcells to increase the ability of a cell type, tissue or tumor to bindangiostatin and inhibit local angiogenesis.

[0144] Angiostatin is effective in treating diseases or processes thatare mediated by, or involve, angiogenesis. The present inventionincludes the method of treating an angiogenesis mediated disease with aneffective amount of angiostatin, or a biologically active fragmentthereof, or combinations of angiostatin fragmetns that collectivelypossess anti-angiogenic activity, or angiostatin agonists andantagonists. The angiogenesis mediated diseases include, but are notlimited to, solid tumors; blood born tumors such as leukemias; tumormetastasis; benign tumors, for example hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis;psoriasis; ocular angiogenic diseases, for example, diabeticretinopathy, retinopathy of prematurity, macular degeneration, cornealgraft rejection, neovascular glaucoma, retrolental fibroplasia,rubeosis; Osler-Webber Syndrome; myocardial angiogenesis; plaqueneovascularization; telangiectasia; hemophiliac joints; angiofibroma;and wound granulation. Angiostatin is useful in the treatment of diseaseof excessive or abnormal stimulation of endothelial cells. Thesediseases include, but are not limited to, intestinal adhesions, Crohn'sdisease, atherosclerosis, scleroderma, and hypertrophic scars, i.e.,keloids. Angiostatin can be used as a birth control agent by preventingvascularization required for embryo implantation. Angiostatin is usefulin the treatment of diseases that have angiogenesis as a pathologicconsequence such as cat scratch disease (Rochele minalia quintosa) andulcers (Helicobacter pylori).

[0145] The synthetic protein fragments of angiostatin have a variety ofuses. The protein that binds to the angiostatin receptor with highspecificity and avidity is radiolabeled and employed for visualizationand quantitation of binding sites using autoradiographic and membranebinding techniques. This application provides important diagnostic andresearch tools. Knowledge of the binding properties of the angiostatinreceptor facilitates investigation of the transduction mechanisms linkedto the receptor.

[0146] In addition, labeling angiostatin proteins with short livedisotopes enables visualization of receptor binding sites in vivo usingpositron emission tomography or other modern radiographic techniques tolocate tumors with angiostatin binding sites.

[0147] Systematic substitution of amino acids within these synthesizedproteins yields high affinity protein agonists and antagonists to theangiostatin receptor that enhance or diminish angiostatin binding to itsreceptor. Such agonists are used to suppress the growth ofmicrometastases, thereby limiting the spread of cancer. Antagonists toangiostatin are applied in situations of inadequate vascularization, toblock the inhibitory effects of angiostatin and promote angiogenesis.For example, this treatment may have therapeutic effects to promotewound healing in diabetics.

[0148] Angiostatin proteins are employed to develop affinity columns forisolation of the angiostatin receptor from cultured tumor cells.Isolation and purification of the angiostatin receptor is followed byamino acid sequencing. Using this information the gene or genes codingfor the angiostatin receptor can be identified and isolated. Next,cloned nucleic acid sequences are developed for insertion into vectorscapable of expressing the receptor. These techniques are well known tothose skilled in the art. Transfection of the nucleic acid sequence(s)coding for angiostatin receptor into tumor cells, and expression of thereceptor by the transfected tumor cells enhances the responsiveness ofthese cells to endogenous or exogenous angiostatin and therebydecreasing the rate of metastatic growth.

[0149] Cytotoxic agents such as ricin, are linked to angiostatin, andhigh affinity angiostatin protein fragments, thereby providing a toolfor destruction of cells that bind angiostatin. These cells may be foundin many locations, including but not limited to, micrometastases andprimary tumors. Proteins linked to cytotoxic agents are infused in amanner designed to maximize delivery to the desired location. Forexample, ricin-linked high affinity angiostatin fragments are deliveredthrough a cannula into vessels supplying the target site or directlyinto the target. Such agents are also delivered in a controlled mannerthrough osmotic pumps coupled to infusion cannulae. A combination ofangiostatin antagonists may be co-applied with stimulators ofangiogenesis to increase vascularization of tissue. This therapeuticregimen provides an effective means of destroying metastatic cancer.

[0150] Angiostatin may be used in combination with other compositionsand procedures for the treatment of diseases. For example, a tumor maybe treated conventionally with surgery, radiation or chemotherapycombined with angiostatin and then angiostatin may be subsequentlyadministered to the patient to extend the dormancy of micrometastasesand to stabilize and inhibit the growth of any residual primary tumor.Additionally, angiostatin, angiostatin fragments, angiostatin antisera,angiostatin receptor agonists, angiostatin receptor antagonists, orcombinations thereof, are combined with pharmaceutically acceptableexcipients, and optionally sustained-release matrix, such asbiodegradable polymers, to form therapeutic compositions.

[0151] A sustained-release matrix, as used herein, is a matrix made ofmaterials, usually polymers, which are degradable by enzymatic oracid/base hydrolysis or by dissolution. Once inserted into the body, thematrix is acted upon by enzymes and body fluids. The sustained-releasematrix desirably is chosen from biocompatible materials such asliposomes, polylactides (polylactic acid), polyglycolide (polymer ofglycolic acid), polylactide co-glycolide (co-polymers of lactic acid andglycolic acid) polyanhydrides, poly(ortho)esters, polyproteins,hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fattyacids, phospholipids, polysaccharides, nucleic acids, polyamino acids,amino acids such as phenylalanine, tyrosine, isoleucine,polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.A preferred biodegradable matrix is a matrix of one of eitherpolylactide, polyglycolide, or polylactide co-glycolide (co-polymers oflactic acid and glycolic acid).

[0152] The angiogenesis-modulating therapeutic composition of thepresent invention may be a solid, liquid or aerosol and may beadministered by any known route of administration. Examples of solidtherapeutic compositions include pills, creams, and implantable dosageunits. The pills may be administered orally, the therapeutic creams maybe administered topically. The implantable dosage unitst may beadministered locally, for example at a tumor site, or which may beimplanted for systemic release of the therapeuticangiogenesis-modulating composition, for example subcutaneously.Examples of liquid composition include formulations adapted forinjection subcutaneously, intravenously, intraarterially, andformulations for topical and intraocular administration. Examples ofaersol formulation include inhaler formulation for administration to thelungs.

[0153] The angiostatin of the present invention also can be used togenerate antibodies that are specific for the inhibitor and itsreceptor. The antibodies can be either polyclonal antibodies ormonoclonal antibodies. These antibodies that specifically bind to theangiostatin or angiostatin receptors can be used in diagnostic methodsand kits that are well known to those of ordinary skill in the art todetect or quantify the angiostatin or angiostatin receptors in a bodyfluid or tissue. Results from these tests can be used to diagnose orpredict the occurrence or recurrence of a cancer and other angiogenicmediated diseases.

[0154] The angiostatin also can be used in a diagnostic method and kitto detect and quantify antibodies capable of binding angiostatin. Thesekits would permit detection of circulating angiostatin antibodies whichindicates the spread of micrometastases in the presence of angiostatinsecreted by primary tumors in situ. Patients that have such circulatinganti-angiostatin antibodies may be more likely to develop multipletumors and cancers, and may be more likely to have recurrences of cancerafter treatments or periods of remission. The Fab fragments of theseanti-angiostatin antibodies may be used as antigens to generateanti-angiostatin Fab-fragment antisera which can be used to neutralizeanti-angiostatin antibodies. Such a method would reduce the removal ofcirculating angiostatin by anti-angiostatin antibodies, therebyeffectively elevating circulating angiostatin levels.

[0155] Another aspect of the present invention is a method of blockingthe action of excess endogenous angiostatin. This can be done bypassively immunizing a human or animal with antibodies specific for theundesired angiostatin in the system. This treatment can be important intreating abnormal ovulation, menstruation and placentation, andvasculogenesis. This provides a useful tool to examine the effects ofangiostatin removal on metastatic processes. The Fab fragment ofangiostatin antibodies contains the binding site for angiostatin. Thisfragment is isolated from angiostatin antibodies using techniques knownto those skilled in the art. The Fab fragments of angiostatin antiseraare used as antigens to generate production of anti-Fab fragment serum.Infusion of this antiserum against the Fab fragments of angiostatinprevents angiostatin from binding to angiostatin antibodies. Therapeuticbenefit is obtained by neutralizing endogenous anti-angiostatinantibodies by blocking the binding of angiostatin to the Fab fragmentsof anti-angiostatin. The net effect of this treatment is to facilitatethe ability of endogenous circulating angiostatin to reach target cells,thereby decreasing the spread of metastases.

[0156] It is to be understood that the present invention is contemplatedto include any derivatives of the angiostatin that have endothelialinhibitory activity. The present invention includes the entireangiostatin protein, derivatives of the angiostatin protein andbiologically-active fragments of the angiostatin protein. These includeproteins with angiostatin activity that have amino acid substitutions orhave sugars or other molecules attached to amino acid functional groups.The present invention also includes genes that code for angiostatin andthe angiostatin receptor, and to proteins that are expressed by thosegenes.

[0157] The proteins and protein fragments with the angiostatin activitydescribed above can be provided as isolated and substantially purifiedproteins and protein fragments in pharmaceutically acceptableformulations using formulation methods known to those of ordinary skillin the art. These formulations can be administered by standard routes.In general, the combinations may be administered by the topical,transdermal, intraperitoneal, intracranial, intracerebroventricular,intracerebral, intravaginal, intrauterine, oral, rectal or parenteral(e.g., intravenous, intraspinal, subcutaneous or intramuscular) route.In addition, the angiostatin may be incorporated into biodegradablepolymers allowing for sustained release of the compound, the polymersbeing implanted in the vicinity of where drug delivery is desired, forexample, at the site of a tumor or implanted so that the angiostatin isslowly released systemically. Osmotic minipumps may also be used toprovide controlled delivery of high concentrations of angiostatinthrough cannulae to the site of interest, such as directly into ametastatic growth or into the vascular supply to that tumor. Thebiodegradable polymers and their use are described, for example, indetail in Brem et al., J. Neurosurg. 74:441-446 (1991), which is herebyincorporated by reference in its entirety.

[0158] The dosage of the angiostatin of the present invention willdepend on the disease state or condition being treated and otherclinical factors such as weight and condition of the human or animal andthe route of administration of the compound. For treating humans oranimals, between approximately 0.5 mg/kilogram to 500 mg/kilogram of theangiostatin can be administered. Depending upon the half-life of theangiostatin in the particular animal or human, the angiostatin can beadministered between several times per day to once a week. It is to beunderstood that the present invention has application for both human andveterinary use. The methods of the present invention contemplate singleas well as multiple administrations, given either simultaneously or overan extended period of time.

[0159] The angiostatin formulations include those suitable for oral,rectal, ophthalmic (including intravitreal or intracameral), nasal,topical (including buccal and sublingual), intrauterine, vaginal orparenteral (including subcutaneous, intraperitoneal, intramuscular,intravenous, intradermal, intracranial, intratracheal, and epidural)administration. The angiostatin formulations may conveniently bepresented in unit dosage form and may be prepared by conventionalpharmaceutical techniques. Such techniques include the step of bringinginto association the active ingredient and the pharmaceutical carrier(s)or excipient(s). In general, the formulations are prepared by uniformlyand intimately bringing into association the active ingredient withliquid carriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0160] Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The formulations may be presented inunit-dose or multi-dose containers, for example, sealed ampules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for example,water for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets of the kind previously described.

[0161] Preferred unit dosage formulations are those containing a dailydose or unit, daily sub-dose, or an appropriate fraction thereof, of theadministered ingredient. It should be understood that in addition to theingredients, particularly mentioned above, the formulations of thepresent invention may include other agents conventional in the arthaving regard to the type of formulation in question. Optionally,cytotoxic agents may be incorporated or otherwise combined withangiostatin proteins, or biologically functional protein fragementsthereof, to provide dual therapy to the patient.

[0162] Angiogenesis inhibiting proteins of the present invention can besynthesized in a standard microchemical facility and purity checked withHPLC and mass spectrophotometry. Methods of protein synthesis, HPLCpurification and mass spectrophotometry are commonly known to thoseskilled in these arts. Angiostatin proteins and angiostatin receptorsproteins are also produced in recombinant E. coli or yeast expressionsystems, and purified with column chromatography.

[0163] Different protein fragments of the intact angiostatin moleculecan be synthesized for use in several applications including, but notlimited to the following; as antigens for the development of specificantisera, as agonists and antagonists active at angiostatin bindingsites, as proteins to be linked to, or used in combination with,cytotoxic agents for targeted killing of cells that bind angiostatin.The amino acid sequences that comprise these proteins are selected onthe basis of their position on the exterior regions of the molecule andare accessible for binding to antisera. The amino and carboxyl terminiof angiostatin, as well as the mid-region of the molecule arerepresented separately among the fragments to be synthesized.

[0164] These protein sequences are compared to known sequences usingprotein sequence databases such as GenBank, Brookhaven Protein,SWISS-PROT, and PIR to determine potential sequence homologies. Thisinformation facilitates elimination of sequences that exhibit a highdegree of sequence homology to other molecules, thereby enhancing thepotential for high specificity in the development of antisera, agonistsand antagonists to angiostatin.

[0165] Angiostatin and angiostatin derived proteins can be coupled toother molecules using standard methods. The amino and carboxyl terminiof angiostatin both contain tyrosine and lysine residues and areisotopically and nonisotopically labeled with many techniques, forexample radiolabeling using conventional techniques (tyrosineresidues-chloramine T, iodogen, lactoperoxidase; lysineresidues-Bolton-Hunter reagent). These coupling techniques are wellknown to those skilled in the art. Alternatively, tyrosine or lysine isadded to fragments that do not have these residues to facilitatelabeling of reactive amino and hydroxyl groups on the protein. Thecoupling technique is chosen on the basis of the functional groupsavailable on the amino acids including, but not limited to amino,sulfhydral, carboxyl, amide, phenol, and imidazole. Various reagentsused to effect these couplings include among others, glutaraldehyde,diazotized benzidine, carbodiimide, and p-benzoquinone.

[0166] Angiostatin proteins are chemically coupled to isotopes, enzymes,carrier proteins, cytotoxic agents, fluorescent molecules,chemiluminescent, bioluminescent and other compounds for a variety ofapplications. The efficiency of the coupling reaction is determinedusing different techniques appropriate for the specific reaction. Forexample, radiolabeling of an angiostatin protein with ¹²⁵I isaccomplished using chloramine T and Na¹²⁵I of high specific activity.The reaction is terminated with sodium metabisulfite and the mixture isdesalted on disposable columns. The labeled protein is eluted from thecolumn and fractions are collected. Aliquots are removed from eachfraction and radioactivity measured in a gamma counter. In this manner,the unreacted Na¹²⁵I is separated from the labeled angiostatin protein.The protein fractions with the highest specific radioactivity are storedfor subsequent use such as analysis of the ability to bind toangiostatin antisera.

[0167] Another application of protein conjugation is for production ofpolyclonal antisera. For example, angiostatin proteins containing lysineresidues are linked to purified bovine serum albumin usingglutaraldehyde. The efficiency of the reaction is determined bymeasuring the incorporation of radiolabeled protein. Unreactedglutaraldehyde and protein are separated by dialysis. The conjugate isstored for subsequent use.

[0168] Antiserum against angiostatin, angiostatin analogs, proteinfragments of angiostatin and the angiostatin receptor can be generated.After protein synthesis and purification, both monoclonal and polyclonalantisera are raised using established techniques known to those skilledin the art. For example, polyclonal antisera may be raised in rabbits,sheep, goats or other animals. Angiostatin proteins conjugated to acarrier molecule such as bovine serum albumin, or angiostatin itself, iscombined with an adjuvant mixture, emulsified and injectedsubcutaneously at multiple sites on the back, neck, flanks, andsometimes in the footpads. Booster injections are made at regularintervals, such as every 2 to 4 weeks. Blood samples are obtained byvenipuncture, for example using the marginal ear veins after dilation,approximately 7 to 10 days after each injection. The blood samples areallowed to clot overnight at 4 C. and are centrifuged at approximately2400×g at 4 C. for about 30 minutes. The serum is removed, aliquoted,and stored at 4 C. for immediate use or at −20 to −90 C. for subsequentanalysis.

[0169] All serum samples from generation of polyclonal antisera or mediasamples from production of monoclonal antisera are analyzed fordetermination of antibody titer. Titer is established through severalmeans, for example, using dot blots and density analysis, and also withprecipitation of radiolabeled protein-antibody complexes using proteinA, secondary antisera cold ethanol or charcoal-dextran followed byactivity measurement with a gamma counter. The highest titer antiseraare also purified on affinity columns which are commercially available.Angiostatin proteins are coupled to the gel in the affinity column.Antiserum samples are passed through the column and anti-angiostatinantibodies remain bound to the column. These antibodies are subsequentlyeluted, collected and evaluated for determination of titer andspecificity.

[0170] The highest titer angiostatin antisera is tested to establish thefollowing; a) optimal antiserum dilution for highest specific binding ofthe antigen and lowest non-specific binding, b) the ability to bindincreasing amounts of angiostatin protein in a standard displacementcurve, c) potential cross-reactivity with related proteins and proteins,including plasminogen and also angiostatin of related species, d)ability to detect angiostatin proteins in extracts of plasma, urine,tissues, and in cell culture media.

[0171] Kits for measurement of angiostatin, and the angiostatinreceptor, are also contemplated as part of the present invention.Antisera that possess the highest titer and specificity and can detectangiostatin proteins in extracts of plasma, urine, tissues, and in cellculture media are further examined to establish easy to use kits forrapid, reliable, sensitive, and specific measurement and localization ofangiostatin. These assay kits include but are not limited to thefollowing techniques; competitive and non-competitive assays,radioimmunoassay, bioluminescence and chemiluminescence assays,fluorometric assays, sandwich assays, immunoradiometric assays, dotblots, enzyme linked assays including ELISA, microtiter plates, antibodycoated strips or dipsticks for rapid monitoring of urine or blood, andimmunocytochemistry. For each kit the range, sensitivity, precision,reliability, specificity and reproducibility of the assay areestablished. Intraassay and interassay variation is established at 20%,50% and 80% points on the standard curves of displacement or activity.

[0172] One example of an assay kit commonly used in research and in theclinic is a radioimmunoassay (RIA) kit. An angiostatin RIA isillustrated below. After successful radioiodination and purification ofangiostatin or an angiostatin protein, the antiserum possessing thehighest titer is added at several dilutions to tubes containing arelatively constant amount of radioactivity, such as 10,000 cpm, in asuitable buffer system. Other tubes contain buffer or preimmune serum todetermine the non-specific binding. After incubation at 4 C. for 24hours, protein A is added and the tubes are vortexed, incubated at roomtemperature for 90 minutes, and centrifuged at approximately 2000-2500×gat 4 C. to precipitate the complexes of antibody bound to labeledantigen.The supernatant is removed by aspiration and the radioactivityin the pellets counted in a gamma counter. The antiserum dilution thatbinds approximately 10 to 40% of the labeled protein after subtractionof the non-specific binding is further characterized.

[0173] Next, a dilution range (approximately 0.1 pg to 10 ng) of theangiostatin protein used for development of the antiserum is evaluatedby adding known amounts of the protein to tubes containing radiolabeledprotein and antiserum. After an additional incubation period, forexample, 24 to 48 hours, protein A is added and the tubes centrifuged,supernatant removed and the radioactivity in the pellet counted. Thedisplacement of the binding of radiolabeled angiostatin protein by theunlabeled angiostatin protein (standard) provides a standard curve.Several concentrations of other angiostatin protein fragments,plasminogen, angiostatin from different species, and homologous proteinsare added to the assay tubes to characterize the specificity of theangiostatin antiserum.

[0174] Extracts of various tissues, including but not limited to primaryand secondary tumors, Lewis lung carcinoma, cultures of angiostatinproducing cells, placenta, uterus, and other tissues such as brain,liver, and intestine, are prepared using extraction techniques that havebeen successfully employed to extract angiostatin. After lyophilizationor Speed Vac of the tisssue extracts, assay buffer is added anddifferent aliquots are placed into the RIA tubes. Extracts of knownangiostatin producing cells produce displacement curves that areparallel to the standard curve, whereas extracts of tissues that do notproduce angiostatin do not displace radiolabeled angiostatin from theangiostatin antiserum. In addition, extracts of urine, plasma, andcerebrospinal fluid from animals with Lewis lung carcinoma are added tothe assay tubes in increasing amounts. Parallel displacement curvesindicate the utility of the angiostatin assay to measure angiostatin intissues and body fluids.

[0175] Tissue extracts that contain angiostatin are additionallycharacterized by subjecting aliquots to reverse phase HPLC. Eluatefractions are collected, dried in Speed Vac, reconstituted in RIA bufferand analyzed in the angiostatin RIA. The maximal amount of angiostatinimmunoreactivity is located in the fractions corresponding to theelution position of angiostatin.

[0176] The assay kit provides instructions, antiserum, angiostatin orangiostatin protein, and possibly radiolabeled angiostatin and/orreagents for precipitation of bound angiostatin-angiostatin antibodycomplexes. The kit is useful for the measurement of angiostatin inbiological fluids and tissue extracts of animals and humans with andwithout tumors.

[0177] Another kit is used for localization of angiostatin in tissuesand cells. This angiostatin immunohistochemistry kit providesinstructions, angiostatin antiserum, and possibly blocking serum andsecondary antiserum linked to a fluorescent molecule such as fluoresceinisothiocyanate, or to some other reagent used to visualize the primaryantiserum. Immunohistochemistry techniques are well known to thoseskilled in the art. This angiostatin immunohistochemistry kit permitslocalization of angiostatin in tissue sections and cultured cells usingboth light and electron microscopy. It is used for both research andclinical purposes. For example, tumors are biopsied or collected andtissue sections cut with a microtome to examine sites of angiostatinproduction. Such information is useful for diagnostic and possiblytherapeutic purposes in the detection and treatment of cancer. Anothermethod to visualize sites of angiostatin biosynthesis involvesradiolabeling nucleic acids for use in in situ hybridization to probefor angiostatin messenger RNA. Similarly, the angiostatin receptor canbe localized, visualized and quantitated with immunohistochemistrytechniques.

[0178] This invention is further illustrated by the following examples,which are not to be construed in any way as imposing limitations uponthe scope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE 1

[0179] Choice of an Animal-Tumor System in Which Growth of Metastasis isInhibited by the Primary Tumor and is Accelerated After Removal of thePrimary Tumor.

[0180] By screening a variety of murine tumors capable of inhibitingtheir own metastases, a Lewis lung carcinoma was selected in which theprimary tumor most efficiently inhibited lung metastasis. SyngeneicC57BI6/J six-week-old male mice were injected (subcutaneous dorsum) with1×10⁶ tumor cells. Visible tumors first appeared after 3-4 days. Whentumors were approximately 1500 mm³ in size, mice were randomized intotwo groups. The primary tumor was completely excised in the first groupand left intact in the second group after a sham operation. Althoughtumors from 500 mm³ to 3000 mm³ inhibited growth of metastases, 1500 mm³was the largest primary tumor that could be safely resected with highsurvival and no local recurrence.

[0181] After 21 days, all mice were sacrificed and autopsied. In micewith an intact primary tumor, there were four +2 visible metastases,compared to fifty +5 metastases in the mice in which the tumor had beenremoved (p<0.0001). These data were confirmed by lung weight, whichcorrelates closely with tumor burden, as has been previouslydemonstrated. There was a 400% increase in wet lung weight in the micethat had their tumors removed compared to mice in which the tumorremained intact (p<0.0001).

[0182] This experimental model gave reproducible data and the experimentdescribed is reproducible. This tumor is labeled “Lewis lungcarcinoma-low metastatic” (LLC-Low). The tumor also suppressedmetastases in a nearly identical pattern in SCID mice, which aredeficient in both B and T lymphocytes.

EXAMPLE 2

[0183] Isolation of a Variant of Lewis Lung Carcinoma Tumor that isHighly Metastatic, Whether or Not the Primary Tumor is Removed.

[0184] A highly metastatic variant of Lewis lung carcinoma arosespontaneously from the LLC-Low cell line of Example 1 in one group ofmice and has been isolated according to the methods described in Example1 and repeatedly transplanted. This tumor (LLC-High) forms more than 30visible lung metastases whether or not the primary tumor is present.

EXAMPLE 3

[0185] Size of Metastases and Proliferation Rate of Tumor Cells WithinThem. Effect of the Primary Tumor that Inhibits Metastases (LLC-Low).

[0186] C57BI6/J mice were used in all experiments. Mice were inoculatedsubcutaneously with LLC-Low cells, and 14 days later the primary tumorwas removed in half of the mice. At 5, 10 and 15 days after the tumorhad been removed, mice were sacrificed. Histological sections of lungmetastases were obtained. Mice with an intact primary tumor hadmicrometastases in the lung which were not neovascularized. Thesemetastases were restricted to a diameter of 12-15 cell layers and didnot show a significant size increase even 15 days after tumor removal.In contrast, animals from which the primary tumor was removed, revealedlarge vascularized metastases as early as 5 days after operation. Thesemetastases underwent a further 4-fold increase in volume by the 15th dayafter the tumor was removed (as reflected by lung weight and histology).Approximately 50% of the animals who had a primary tumor removed died oflung metastases before the end of the experiment. All animals with anintact primary tumor survived to the end of the experiment.

[0187] Replication rate of tumor cells within metastases was determinedby counting nuclei stained with BrdU which had been previously injectedinto the mice. The high percentage of tumor cells incorporating BrdU insmall, avascular metastases of animals with an intact primary tumor wasequivalent to the BrdU incorporation of tumor cells in the largevascularized metastases of mice from which the primary tumor had beenremoved (FIG. 3). This finding suggests that the presence of a primarytumor has no direct effect on the replication rate of tumor cells withina metastasis.

[0188] In FIG. 3, the left panel shows BrdU labeling index of tumorcells in the lung in the presence or absence of a primary tumor. Beforeimmunohistochemical staining, sections were permeabilized with 0.2 M HClfor 10 minutes and digested with 1 μg/ml proteinase K (BoehringerMannheim GmbH, Mannheim, Germany) in 0.2 M Tris-HCl, 2 mM CaCl₂ at 37°C. for 15 minutes. Labeling index was estimated by counting percentageof positive nuclei at 250 power. The right panel of FIG. 3 depicts ananalysis of total lung weight of tumors with primary tumors intact orremoved 5, 10 and 15 days after operation. Animals were sacrificed 6hours after intraperitoneal injection of BrdU (0.75 mg/mouse).

EXAMPLE 4

[0189] Inhibition of Angiogenesis in Lung Metastases in the Presence ofan Intact Primary Tumor.

[0190] To measure the degree of vascularization in lung metastases,tissues were stained with antibodies against von Willebrand factor (anendothelial specific marker, available from Dako Inc., Carpenteria,Calif.). Metastases from animals with intact tumors formed a thin cuff(8-12 tumor cell layers) around existing pulmonary vessels. Except forthe endothelial cells of the vessel lining, no or few cells werepositive for von Willebrand factor. In contrast, lung metastases ofanimals 5 days after removal of the primary tumor were not only largerbut were also infiltrated with capillary sprouts containing endothelialcells which stained strongly for von Willebrand factor.

[0191] In immunohistochemical analysis of the presence of endothelialcells in lung metastases, a lung metastasis with the primary lung tumorintact 19 days after inoculation, had a cuff of tumor cells around apre-existing microvessel in the lung. The metastasis was limited to 8 to12 cell layers. There was no evidence of neovascularization around themicrovessel, and it did not contain any new microvessels. This wastypical of the maximum size of an avascular pre-angiogenic metastasis.

[0192] In an immunohistochemical analysis of tissue collected five daysafter the primary tumor was resected (19 days after inoculation of theprimary tumor), the metastasis surrounded a pre-existing vessel in thelung. In contrast, in the sample where the primary tumor was notresected, the tumor was neovascularized. Thus, an intact primary tumorinhibits formation of new capillary blood vessels in metastases, butproliferation of tumor cells within a metastasis are not affected by theprimary tumor.

EXAMPLE 5

[0193] A Primary Tumor Inhibits Angiogenesis of a Second Tumor Implantedin the Mouse Cornea. Growth of this Second Tumor is Inhibited.

[0194] A 0.25 to 0.5 mm² Lewis lung tumor (LLC-Low) was implanted in themouse cornea on day 0. (Muthukkaruppan Vr., et al., Angiogenesis in themouse cornea. Science 205:1416-1418, 1979) A primary tumor was formed byinoculating 1×10⁶ LLC-Low cells subcutaneously in the dorsum, either 4or 7 days before the corneal implant; or on the day of the cornealimplant; or 4 or 7 days after the corneal implant. Control mice receivedthe corneal implant but not the subcutaneous tumor. Other control micereceived the corneal implant and an inoculation of LLC-High tumor cellsin the dorsum 4 days before the corneal implant. The corneas wereevaluated daily by slit-lamp stereomicroscopy for the growth of thecorneal tumor (measured by an ocular micrometer) and for the growth ofnew capillary vessels from the edge of the corneal limbus.

[0195] In control mice not bearing a primary subcutaneous tumor, amajority of corneas (6/8) developed neovascularization starting at day 6to 7 days after corneal implantation and continuing to day 10. By day10, the vascularized corneal tumors had reached approximately a quarterof the volume of the whole eye. In the presence of the primarysubcutaneous LLC-Low tumor, the corneal implants did not becomevascularized if the primary tumor was in place by at least 4 days ormore before the corneal implant (Table 1). In the absence ofneovascularization, corneal tumors grew slowly as thin, white, avasculardiscs within the cornea.

[0196] However, if the primary tumor was not implanted until 4 daysafter the corneal implant, corneas became vascularized and 3/3 cornealtumors grew at similar rates as the non-tumor bearing controls. In thepresence of the primary subcutaneous LLC-High tumor, the majority ofcorneas (2/3) developed neovascularization starting at day 7 aftercorneal implantation and continuing to day 10. By day 10, thevascularized corneal tumors again had reached approximately a quarter ofthe volume of the whole eye. TABLE 1 Inhibition of tumor angiogenesis inthe cornea by a primary subcutaneous tumor. [All primary tumors areLLC-Low except (*) which is LLC-High]. Day of eye implant 0 0 0 0 0 0 0Day of primary tumor −7 −4 −4* 0 none +4 +7 implant Number of mice withnew 2/10 0/9 2/3 2/3 6/8 3/3 2/3 corneal vessels at day 10

EXAMPLE 6

[0197] Primary Intact Tumor Inhibits Angiogenesis Induced by a SecondarySubcutaneous Implant of Basic Fibroblast Growth Factor (bFGF.).

[0198] Although the experiments described in Examples V and VI show thata primary tumor inhibits angiogenesis in a secondary metastasis, thesestudies do not reveal whether the primary tumor: (i) inhibitsendothelial proliferation (or angiogenesis) directly, or (ii) indirectlyby down-regulating the angiogenic activity of the metastatic tumorcells. To distinguish between these two possibilities, a focus ofsubcutaneous angiogenesis was induced by an implant of matrigelcontaining basic fibroblast growth factor (bFGF). (Passaniti A, et al.,A simple, quantitative method for assessing angiogenesis andanti-angiogenic agents using reconstituted basement membrane, heparinand fibroblast growth factor. Lab. Invest. 67:519, 1992)

[0199] Matrigel (an extract of basement membrane proteins), containingeither 25 or 50 ng/ml bFGF in the presence of heparin, was injectedsubcutaneously on the ventral surface of normal and tumor-bearing mice(LLC-Low). Mice were sacrificed 4 days later and hemoglobinconcentration in the gel was measured to quantify blood vesselformation. It has previously been shown that the number of new vesselswhich enter the matrigel is correlated with hemoglobin concentration.(Folkman J., Angiogenesis and its inhibitors in “Important Advances inOncology 1985”, V T DeVita, S. Hellman and S. Rosenberg, editors, J. B.Lippincott, Philadelphia 1985) Some gels were also prepared forhistological examination. In normal mice, matrigel pellets whichcontained 50 ng/ml bFGF were completely red. They were heavily invadedby new capillary vessels, and contained 2.4 g/dl hemoglobin. Matrigelwhich lacked bFGF was translucent and gray and contained only 0.4 g/dlhemoglobin (a 6-fold difference). In contrast, matrigel from mice with aprimary tumor contained only 0.5 g/dl (FIG. 4).

[0200] The near complete inhibition of angiogenesis in this experimentsuggests that the presence of a Lewis lung primary tumor can inhibitbFGF-induced angiogenesis directly.

EXAMPLE 7

[0201] Transfer of Serum from a Tumor-Bearing Animal to an Animal fromWhich the Primary Tumor Has Been Removed Suppresses Metastases.

[0202] Mice were implanted with Lewis lung carcinoma as described above.After 15 days, when tumors were approximately 1500 mm³, the mice wererandomized into four groups. Three groups underwent complete surgicalresection of the primary tumor; in one group the tumors were left inplace (after a sham surgical procedure). The mice in the three resectiongroups then received daily intraperitoneal injections of saline, serumfrom normal nontumor bearing mice, or serum from mice with 1500 mm³Lewis lung carcinomas. The group of mice with the tumors left intactreceived intraperitoneal saline injections. All mice were treated for 21days, after which the animals were euthanized and lung metastases werecounted (Table 2). TABLE 2 Primary Tumor Primary Tumor Removed IntactTreatment Saline Serum from Serum from Saline (Intraperitoneal normalmice tumor-beaiing Injections Injections) mice Number of Lung 55 ± 5 50± 4 7 ± 2 3 ± 1 Metastases:

[0203] These results were confirmed by lung weight. p=<0.0001 for thedifference between the two groups [(55 & 50) vs. (7 & 3)]. Similarresults have been obtained using angiostatin from the urine oftumor-bearing animals.

EXAMPLE 8

[0204] Bovine Capillary Endothelial (BCE) Cell Assay

[0205] BCE cells are used between passages 9 and 14 only. At day 0, BCEcells are plated onto gelatinized (1.5% gelatin in PBS at 37°, 10% CO₂for 24 hours and then rinsed with 0.5 ml PBS) 24 well plates at aconcentration of 12,500 cells/well. Cell counts are performed using ahemocytometer. Cells are plated in 500 μl DMEM with 10% heat-inactivated(56° C. for 20 minutes) bovine calf serum and 1% glutamine-pen-strep(GPS).

[0206] BCE cells are challenged-as follows: Media is removed andreplaced with 250 μl of DMEM/5% BCS/1%GPS. The sample to be tested isthen added to wells. (The amount varies depending on the sample beingtested) Plates are placed at 37° C./10% CO₂ for approximately 10minutes. 250 μl of DMEM/5% BCS/1% GPS with 2 ng/ml bFGF is added to eachwell. The final media is 500 μl of DMEM/5% BCS/1%GPS/with 1 ng/ml bFGF.The plate is returned to 37° C./10% CO₂ incubator for 72 hours.

[0207] At day 4, cells are counted by removing the medium and thentrypsinizing all wells (0.5 ml trypsin/EDTA) for 2 to 3 minutes. Thesuspended cells are then transferred to scintillation vials with 9.5 mlHemetall and counted using a Coulter counter. A unit of activity is thatamount of serum containing angiostatin that is capable of producinghalf-maximal inhibition of capillary endothelial proliferation whenendothelial cells are incubated in bFGF 1 ng/ml for 72 hours.

EXAMPLE 9

[0208] Serum from Mice Bearing the Low Metastatic Lewis Lung Tumor(LLC-Low) Inhibits Capillary Endothelial Cell Proliferation in Vitro.

[0209] Bovine capillary endothelial cells were stimulated by basicfibroblast growth factor (bFGF 1 ng/ml), in a 72-hour proliferationassay. The serum of tumor-bearing mice added to these cultures inhibitedendothelial cell proliferation in a dose-dependent and reversiblemanner. Normal serum was not inhibitory (FIG. 5). Endothelial cellproliferation was inhibited in a similar manner (relative to controls)by serum obtained from tumor-bearing nu/nu mice and SCID mice. After theprimary tumor was removed, angiostatin activity disappeared from theserum by 3-5 days.

[0210] Tumor-bearing serum also inhibited bovine aortic endothelialcells and endothelial cells derived from a spontaneous mousehemangioendothelioma, (Obeso, et al., “Methods in LaboratoryInvestigation, A Hemangioendothelioma-derived cell line; Its use as aModel for the Study of Endothelial Cell Biology,” Lab Invest., 63(2),pgs 259-269, 1990) but did not inhibit Lewis lung tumor cells, 3T3fibroblasts, aortic smooth muscle cells, mink lung epithelium, or W138human fetal lung fibroblasts.

EXAMPLE 10

[0211] Serum from Mice Bearing the Lewis Lung Tumor (LLC-High) that DoesNot Inhibit Metastases, Does Not Inhibit Capillary Endothelial CellProliferation in Vitro.

[0212] Serum from mice bearing a primary tumor of the LLC-High did notsignificantly inhibit proliferation of bFGF-stimulated bovine capillaryendothelial cells relative to controls. Also, when this serum wassubjected to the first two steps of purification (heparin-Sepharosechromatography and gel filtration), angiostatin activity was not foundin any fractions.

EXAMPLE 11

[0213] Ascites from Lewis Lung Carcinoma (Low Metastatic), AlsoGenerates Angiostatin Serum.

[0214] Mice received intraperitoneal injections of either LLC-Low orLLC-High tumor cells (10⁶), and one week later, 1-2 ml of bloody asciteswas obtained from each of 10-20 mice. Mesenteric tumor seeding was seen.The mice were then euthanized. Serum was obtained by cardiac puncture.Serum was also obtained from normal, non-tumor-bearing mice as acontrol. Serum and ascites were centrifuged to remove cells, and thesupernate was assayed on bovine capillary endothelial cells stimulatedby bFGF (1 ng/ml) (see Example IX). Ascites originating from both tumortypes stimulated significant proliferation of capillary endothelialcells (e.g., 100% proliferation) over controls after 72 hours (FIG. 6).In contrast, serum from the low metastatic mice inhibited endothelialcell proliferation (inhibition to 79% of controls). The serum from thehigh metastatic line was stimulatory by 200%.

[0215] These data show that the ascites of the low metastatic linecontains a predominance of endothelial growth stimulator overangiostatin. This condition is analogous to a solid primary tumor.Furthermore, angiostatin activity appears in the serum, as though itwere unopposed by stimulatory activity. This pattern is similar to thesolid primary tumor (LLC-Low). The ascites from the high metastatictumor (LLC-High) also appears to contain a predominance of endothelialcell stimulator, but angiostatin cannot be identified in the serum.

EXAMPLE 12

[0216] Fractionation of Angiostatin from Serum by Column Chromatographyand Analysis of Growth-Inhibitory Fractions by SDS-PAGE.

[0217] To purify the angiostatin(s), serum was pooled from tumor-bearingmice. The inhibitory activity, assayed according the above-described invitro inhibitor activity assay, was sequentially chromatographed usingheparin-Sepharose, Biogel AO.5 mm agarose, and several cycles ofC4-reverse phase high performance liquid chromatography (HPLC). SDS-PAGEof the HPLC fraction which contained endothelial inhibitory activity,revealed a discrete band of apparent reduced M_(r) of 38,000 Daltons,which was purified approximately 1 million-fold (see Table 3) to aspecific activity of approximately 2×10⁷. At different stages of thepurification, pooled fractions were tested with specific antibodies forthe presence of known endothelial inhibitors. Platelet factor-4,thrombospondin, or transforming growth factor beta, were not found inthe partially purified or purified fractions. TABLE 3 Specific activity(units*/mg) Fold purification Serum 1.69 1 Heparin Sepharose 14.92 8.8Bio-gel AO.5 m 69.96 41.4 HPLC/C4 2 × 10⁷ 1.2 × 10⁶

EXAMPLE 13

[0218] Fractionation of Angiostatin from Urine by Column Chromatographyand Analysis of Growth-Inhibitory Fractions by SDS-PAGE.

[0219] Purification of the endothelial cell inhibitor(s) from serum ishampered by the small volume of serum that can be obtained from eachmouse and by the large amount of protein in the serum.

[0220] Urine from tumor bearing mice was analyzed and found that itcontains an inhibitor of endothelial cell proliferation that is absentfrom the urine of non-tumor bearing mice and from mice with LLC-hightumors. Purification of the endothelial cell inhibitory activity wascarried out by the same strategy that was employed for purification ofserum (described above) (FIG. 7).

[0221]FIG. 7 shows C4 reverse phase chromatography of partially purifiedserum or urine from tumor-bearing animals. All fractions were assayed onbovine capillary endothelial cells with bFGF in a 72-hour proliferationassay as described in Example IX. A discrete peak of inhibition was seenin both cases eluting at 30-35% acetonitrile in fraction 23.SDS-polyacrylamide gel electrophoresis of inhibitory fraction from thethird cycle of C4 reverse phase chromatography of serum fromtumor-bearing animals showed a single band at about 38,000 Daltons.

EXAMPLE 14

[0222] Characterization of Circulating Angiostatin.

[0223] Endothelial inhibition was assayed according to the proceduredescribed in Example 9. Angiostatin was isolated on a Synchropak HPLC C4column. (Synchrom, Inc. Lafayette, Ind.) The inhibitor was eluted at 30to 35% acetonitrile gradient. On a sodium dodecyl sulfate polyacrylamidegel electrophoresis (PAGE) gel under reducing conditions(b-mercaptoethanol(5% v/v), the protein band with activityeluted at 38kilodaltons. Under non-reducing conditions, the protein with activityeluted at 28 kilodaltons. The activity is found at similar pointswhether the initial sample was isolated from urine or from serum.Activity was not detected with any other bands.

[0224] Activity associated with the bands was lost when heated (100° C.for 10 minutes) or treated with trypsin. When the band with activity wasextracted with a water/chloroform mixture (1:1), the activity was foundin the aqueous phase only.

EXAMPLE 15

[0225] Purification of Inhibitory Fragments from Human Plasminogen:

[0226] Plasminogen lysine binding site I was obtained from SigmaChemical Company. The preparation is purified human plasminogen afterdigestion with elastase. Lysine binding site I obtained in this manneris a population of proteins that contain, in aggregate, at least thefirst three triple-loop structures (numbers 1 through 3) in the plasminA-chain (Kringle 1+2+3). (Sotrrup-Jensen, L., et al. in Progress inChemical Fibrinolysis and Thrombolysis, Vol. 3, 191, Davidson, J. F., etal. eds. Raven Press, New York 1978 and Wiman, B., et al., Biochemica etBiophysica Acta, 579, 142 (1979)). Plasminogen lysine binding site I(Sigma Chemical Company, St. Louis, Mo.) was resuspended in water andapplied to a C4-reversed phase column that had been equilibrated withHPLC-grade water/0.1% TFA. The column was eluted with a gradient ofwater/0.1% TFA to acetonitrile/0.1% TFA and fractions were collectedinto polypropylene tubes. An aliquot of each was evaporated in a speedvac, resuspended with water, and applied to BCEs in a proliferationassay. This procedure was repeated two times for the inhibitoryfractions using a similar gradient for elution. The inhibitory activityeluted at 30-35% acetonitrile in the final run of the C4 column.SDS-PAGE of the inhibitory fraction revealed 3 discrete bands ofapparent reduced molecular mass of 40, 42.5, and 45 kd. SDS-PAGE undernon-reducing conditions revealed three bands of molecular mass 30, 32.5,and 35 kd respectively.

EXAMPLE 16

[0227] Extraction of Inhibitory Activity from SDS-PAGE

[0228] Purified inhibitory fractions from human plasminogen basedpurifications were resolved by SDS-PAGE under non-denaturing conditions.Areas of the gel corresponding to bands seen in neighboring lanes loadedwith the same samples by silver staining were cut from the gel andincubated in 1 ml of phosphate buffered saline at 4° C. for 12 hours inpolypropylene tubes. The supernatant was removed and dialyzed twiceagainst saline for 6 hours (MWCO=6-8000) and twice against distilledwater for 6 hours. The dialysate was evaporated by vacuumcentrifugation. The product was resuspended in saline and applied tobovine capillary endothelial cells stimulated by 1 ng/ml basicfibroblast growth factor in a 72 hour assay. Protein extracted from eachof the three bands inhibited the capillary endothelial cells.

EXAMPLE 17

[0229] Plasminogen Fragment Treatment Studies

[0230] Mice were implanted with Lewis lung carcinomas and underwentresections when the tumors were 1500-2000 mm³. On the day of operation,mice were randomized into 6 groups of 6 mice each. The mice receiveddaily intraperitoneal injections with the three purified inhibitoryfragments of human plasminogen, whole human plasminogen, urine fromtumor-bearing animals, urine from normal mice, or saline. One group oftumor-bearing animals that had only a sham procedure was treated withsaline injections. Immediately after removal of the primary tumor, themice receive an intraperitoneal injection of 24 μg (1.2 mg/kg/day/mouse)of the inhibitory plasminogen fragments as a loading dose. They thenreceive a daily intraperitoneal injections of 12 μg of the inhibitoryfragment (0.6 mg/kg/day/mouse) for the duration of the experiment.Control mice receive the same dose of the whole plasminogen moleculeafter tumor removal. For the urine treatments, the urine of normal ortumor bearing mice is filtered, dialyzed extensively, lyophilized, andthen resuspended in sterile water to obtain a 250 fold concentration.The mice are given 0.8 ml of the dialyzed urine concentrate, either fromtumor bearing mice or normal mice, in two intraperitoneal injections onthe day of removal of the primary tumor as a loading dose. They thenreceive daily intraperitoneal injections of 0.4 ml of the dialyzed andconcentrated urine for the course of the experiment. Treatments werecontinued for 13 days at which point all mice were sacrificed andautopsied.

[0231] The results of the experiment are shown in FIGS. 8 and 9. FIG. 8shows surface lung metastases after the 13 day treatment. Surface lungmetastases refers to the number of metastases seen in the lungs of themice at autopsy. A stereomicroscope was used to count the metastases.FIG. 8 shows the mean number of surface lung metastases that was countedand the standard error of the mean. As shown, the group of mice with theprimary tumor present showed no metastases. The mice in which theprimary tumor was resected and were treated with saline showed extensivemetastases. The mice treated with the human derived plasminogen fragmentshowed no metastases. The mice treated with whole plasminogen showedextensive metastases indicating that the whole plasminogen molecule hasno endothelial inhibitory activity. Those mice treated with dialyzed andconcentrated urine from tumor bearing mice showed no metastases. Micetreated with concentrated urine from normal mice showed extensivemetastases. When the weight of the lung was measured, similar resultswere obtained (FIG. 9).

EXAMPLE 18

[0232] Amino Acid Sequence of Murine and Human Angiostatin.

[0233] The amino acid sequence of angiostatin isolated from mouse urineand angiostatin isolated from the human lysine binding site I fragmentpreparation was determined on an Applied Biosystem Model 477A proteinsequencer. Phenylthiohydantoin amino acid fractions were identified withan on-line ABI Model 120A HPLC. The amino acid sequence determined fromthe N-terminal sequence and the tryptic digests of the murine and humanangiostatin indicate that the sequence of the angiostatin is similar tothe sequence beginning at amino acid number 98 of murine plasminogen.Thus, the amino acid sequence of the angiostatin is a moleculecomprising a protein having a molecular weight of between approximately38 kilodaltons and 45 kilodaltons as determined by reducingpolyacrylamide gel electrophoresis and having an amino acid sequencesubstantially similar to that of a murine plasminogen fragment beginningat amino acid number 98 of an intact murine plasminogen molecule. Thebeginning amino acid sequence of the murine angiostatin (SEQ ID NO:2) isshown in FIG. 1. The length of the amino acid sequence may be slightlylonger or shorter than that shown in the FIG. 1.

[0234] N terminal amino acid analysis and tryptic digests of the activefraction of human lysine binding site I (See Example 15) show that thesequence of the fraction begins at approximately amino acid 97 or 99 ofhuman plasminogen and the human angiostatin is homologous with themurine angiostatin. The beginning amino acid sequence of the humanangiostatin (starting at amino acid 98) is shown in FIG. 2, (SEQ IDNO:3). The amino acid sequence of murine and human angiostatin iscompared in FIG. 2 to corresponding internal amino acid sequences fromplasminogen of other species including porcine, bovine, and Rhesusmonkey plasminogen, indicating the presence of angiostatin in thosespecies.

EXAMPLE 19

[0235] Expression of Human Angiostatin in E. coli.

[0236] The pTrcHisA vector (Invitrogen) (FIG. 10) was used to obtainhigh-level, regulated transcription from the trc promoter for enhancedtranslation efficiency of eukaryotic genes in E. coli. Angiostatin isexpressed fused to an N-terminal nickel-binding poly-histidine tail forone-step purification using metal affinity resins. The enterokinasecleavage recognition site in the fusion protein allows for subsequentremoval of the N-terminal histidine fusion protein from the purifiedrecombinant protein. The recombinant human angioststin protein was foundto bind lysine; is cross-reactive with monoclonal antibodies specificfor kringle regions 1, 2 and 3, and inhibits bFGF-driven endothelialcell proliferation in vitro.

[0237] To construct the insert, the gene fragment encoding humanangiostatin is obtained from human liver mRNA which is reversetranscribed and amplified using the polymerase chain reaction (PCR) andspecific primers. The product of 1131 base pairs encodes amino acids 93to 470 of human plasminogen. The amplified fragment was cloned into theXhoI/KpnI site of pTrcHisA, and the resultant construct transformed intoXL-1B (available from Stratagene) E. coli host cells. A control clonecontaining the plasmid vector pTrcHisA alone was transformed inot XL-1BE. coli host cells as well. This clone is referred to as the vectorcontrol clone. Both clones were purified identically as described below.

[0238] Expressing colonies were selected in the following manner. Colonylifts of E. coli transformed with the gene encoding angiostatin weregrown on IPTG impregnated nitrocellulose filters and overlaid on an LBagar plate. Following IPTG induction of expression, colonies were lysedon nitrocellulose filters. The nitrocellulose lifts were blocked, rinsedand probed with two separate monoclonal antibodies (mAbs Dcd and Vap;gift of S. G. McCance and F. J. Castellino, University of Notre Dame)which recognize specific conformations of angiostatin. Stronglyexpressing colonies recognized by the mAbs were selected.

[0239] To identify the optimal time for maximal expression, cells werecollected at various times before and after IPTG induction and exposedto repeated freeze-thaw cycles, followed by analysis with SDS-PAGE,immunoblotting and probing with mAbs Dcd and Vap.

[0240] From these, clone pTrcHisA/HAsH4 was selected. Induction withIPTG was for 4 hours after which the cell pellet was collected andresuspended in 50 mM Tris pH 8.0, 2 mM EDTA, 5% glycerol and 200 mg/mllysozyme and stirred for 30 min. at 4° C. The slurry was centrifuged at14,000 rpm for 25 min. and the pellet resuspended in 50 mM Tris pH 8.0,2 mM EDTA, 5% glycerol and 0.1% DOC. This suspension was stirred for 1hr. at 4° C., and then centrifuged at 14,000 rpm for 25 min. Thesupernatant fraction at this step contains expressed angiostatin. The E.coli expressed human angiostatin was found to possess the physicalproperty of native angiostatin, that is the ability to bind lysine. TheE. coli expressed angiostatin was thus purified over a lysine-sepharose(Pharmacia or Sigma) column in a single step. Elution of angiostatinfrom the column was with 0.2M epsilon-amino-n-caproic acid pH7.5.

[0241] Subsequent to these experiments, scale-up 10 L fermentationbatches of clone pTrcHisA/HAsH4 was performed. The cells obtained fromthis scaled-up induction were pelleted and resuspended in50 mM TrispH7.5, cracked at 10,000 psi thrice chilling at 10° C. in-betweenpasses. The lysate obtained was clarified by centrifugation at 10,000rpm for 30 min at 4° C., and expressed angiostatin isolated overlysine-sepharose (FIG. 11).

[0242] Purified E. coli expressed human angiostatin was dialysedexhaustively against water and lyophilized. The expressed humanangiostatin was resuspended in media (DMEM, 5% BCS, 1%Gentamycin/penicillin/streptomycin) to an estimated concentration of 3ug/ml, and used in bovine capillary endothelial (BCE) cell assays invitro, as described in EXAMPLE 8, pg.39. Similarly, the control clonecontaining the vector alone was treated in the identical fashion as theclone pTrcHisA/HAsH4. It was induced with IPTG identically, and thebacterial lysate used to bind lysine, eluted with 0.2 M amino caproicacid, dialysed exhaustively and lyophilized. This control preparationwas resuspended in media also at an estimated concentration of 3 ug/ml.The samples of recombinant angiostatin, and controls were obtained fromdifferent induction and fermentation batches as well as seperatepurification runs, and were all coded at EntreMed, Maryland. BCE assayswere performed with these coded samples in a blinded fashion atChildren's Hospital, Boston.

[0243] The results of BCE assays of recombinant human angiostatin showedthat human angiostatin expressed in E.coli inhibited the proliferationof BCE cells due to bFGF (used at 1 ng/ml) (FIG. 12). The stockrecombinant angiostatin in media (at about 3 ug/ml) was used at a 1:5,1:10 and 1:20 dilution. Percent inhibition was calculated as follows:$1 - \frac{{{number}\quad {of}\quad {cells}\quad {with}\quad {angiostatin}}\quad - {{number}\quad {of}\quad {cells}\quad {at}\quad {day}\quad 0}}{{{number}\quad {of}\quad {cells}\quad {with}\quad {bFGF}\quad {alone}} - {{number}\quad {of}\quad {cells}\quad {at}\quad {day}\quad 0}}$

[0244] The percent inhibition of BCE cell proliferation was comparableor higher to that of plasminogen derived angiostatin at similarconcentrations. The results from a repeat run of the BCE assay aredepicted in FIG. 13, where at a 1:5 dilution of the stock, recombinantangiostatin gave similar percent inhibitions to those obtained withplasminogen derived angiostatin. FIG. 13 shows the surprising resultthat human recombinant angiostatin protein inhibits over 60%, and asmuch as over 75% of BCE proliferation in culture.

EXAMPLE 20

[0245] Angiostatin Maintains Dormancy of Micrometastases by Increasingthe Rate of Apoptosis.

[0246] Following subcutaneous inoculation of C57 BL6/J mice with Lewislung carcinoma cells (1×10⁶), primary tumors of approximately 1.5 cm³developed. Animals were subject to either surgical removal of theprimary tumor or sham surgery. At 5, 10 and 15 days after surgery, micewere sacrificed and their lungs prepared for histological examination.Animals with resected primary tumors showed massive proliferation ofmicrometastases compared to sham operated controls (FIG. 14). Thesechanges were accompanied by a significant increase in lung weight.

[0247] Analysis of tumor cell proliferation, as measured by uptake ofbromo-deoxyuridine (BrdU) showed no differences between animals withintact primary tumors or resected tumors at 5, 9 and 13 days, indicatingthat the increase in tumor mass could not be explained by increasedproliferation (FIG. 15). Accordingly, cell death was examined in theseanimals. Apoptosis, a process of cell death that is dependent on changesin gene expression and accounts for elimination of cells duringdevelopment and in rapidly proliferating tissues such as the smallintestine, was examined by immunohistochemically labeling fragmented DNAwith the terminal deoxynucleotidyl transferase (TdT) technique. Theapoptotic index was determined at each time of sacrifice. The removal ofprimary tumors caused a statistically significant increase(approximately 3 to 4 fold) in the apoptotic index at all times examined(FIG. 15).

[0248] Supporting evidence was obtained by treating mice with removedprimary tumors with an exogenous suppressor of angiogenesis. Thissubstance, TNP-1470 (O-chloroacetylcarbamoyl fumagillol, previouslynamed AGM-1470), is an analogue of fumagillin with reportedanti-angiogenic activity. Subcutaneous injection of TNP-1470 (30 mg/kgevery two days) produced results that were strikingly similar to thosedescribed above for animals that had intact primary tumors. Theseanimals displayed a lower lung weight, equivalent proliferative indexand increased apoptotic index compared to saline-injected controls (FIG.16).

[0249] These data indicate that metastases remain dormant when tumorcell proliferation is balanced by an equivalent rate of cell death. Theremoval of the primary tumor causes a rapid increase in the growth ofmetastases, probably due to the removal of angiogenesis inhibitors(angiostatin) which control metastatic growth by increasing apoptosis intumor cells. These effects are similar to those seen following removalof primary tumors and administration of an exogenous inhibitor ofangiogenesis. Taken together, these data suggest that the primary tumorreleases angiostatin which maintains dormancy of micrometastases.

EXAMPLE 21

[0250] Treatment of Primary Tumors with Angiostatin in Vivo.

[0251] Angiostatin was purified from human plasminogen by limitedelastase digestion as described in Example 15 above. Angiostatin wasresuspended in phosphate-buffered saline for administration into sixweek old male C57BI6/J mice. Animals were implanted subcutaneously with1×10⁶ tumor cells of either the Lewis lung carcinoma or T241fibrosarcoma. Treatment with angiostatin is begun after four days whentumors are 80-160 mm³ in size. Mice received angiostatin injections ineither a single injection of 40 mg/kg or two 80 mg/kg injections viaintraperitoneal (ip) or subcutaneous (sc) routes. Animals weresacrificed at various times after treatment extending to 19 days.

[0252] Angiostatin, administered at a daily dose of 40 mg/kg ip,produced a highly significant inhibition of the growth of T241 primarytumors (FIG. 17). This inhibitory effect on growth was visibly evidentwithin 2 days and increased in magnitude throughout the time course ofthe study. By day 18, angiostatin-treated mice had tumors that wereapproximately 38% of the volume of the saline injected controls. Thisdifference was statistically significant (p<0.001, Students t-test).

[0253] Angiostatin treatment (total dose of 80 mg/kg/day, administeredtwice daily at 40 mg/kg ip or sc) also significantly reduced the growthrate of LLC-LM primary tumors (FIG. 17). This inhibitory effect wasevident at 4 days and increased in magnitude at all subsequent timesexamined. On the last day of the experiment (day 19),angiostatin-treated mice possessed a mean tumor volume that was only 20%of the saline-injected controls which was significantly different(p<0.001 Students t-test).

[0254] In another series of experiments angiostatin was administered (50mg/kg q12 h) to mice implanted with T241 fibrosarcoma, Lewis lungcarcinoma (LM) or reticulum cell sarcoma cells. For each tumor celltype, the mice receiving angiostatin had substantially reduced tumorsize. FIG. 19 demonstrates that for T241 fibrosarcoma, the angiostatintreated mice had mean tumor volumes that were only 15% of the untreatedmice at day 24. FIG. 20 demonstrates that for Lewis lung carcinoma (LM),the angiostatin treated mice had mean tumor volumes that were only 13%of the untreated mice at day 24. FIG. 21 demonstrates that for reticulumsacroma, the angiostatin treated mice had mean tumor volumes that wereonly 19% of the untreated mice at day 24. The data represent the averageof 4 mice at each time point.

[0255] These results demonstrate that angiostatin is an extremely potentinhibitor of the growth of three different primary tumors in vivo.

EXAMPLE 22

[0256] Treatment of Human Cell-Derived Primary Tumors in Mice withAngiostatin in Vivo.

[0257] The effect of angiostatin on two human tumor cell lines, humanprostate carcinoma PC-3 and human breast carcinoma MDA-MB, was studied.Immunodeficient SCID mice were implanted with human tumor cells, and themice treated with 50 mg/kg angiostatin every 12 hours essentially asdescribed in Example 21. The results demonstrate that the angiostatinprotein of the present invention is a potent inhibitor of human tumorcell growth. FIG. 22 shows that for human prostate carcinoma PC-3, theangiostatin treated mice had only 2% of the mean tumor volume comparedto the untreated control mice at day 24. FIG. 23 shows that for humanbreast carcinoma MDA-MB, he angiostatin treated mice had only 8% of themean tumor volume compared to the untreated control mice at day 24.

EXAMPLE 23

[0258] Gene Therapy—Effect of Transfection of the Angiostatin Gene onTumor Volume.

[0259] A 1380 base pair DNA sequence for angiostatin derived from mouseplasminogen cDNA (obtained from American Type Culture Collection(ATCC)), coding for mouse plasminogen amino acids 1-460, was generatedusing PCR and inserted into an expression vector. The expression vectorwas transfected into T241 fibrosarcoma cells and the transfected cellswere implanted into mice. Control mice received either non-transfectedT241 cells, or T241 cells transfected with the vector only (i.e.non-angiostatin expressing transfected cells). Threeangiostatin-expressing transfected cell clones were used in theexperiment. Mean tumor volume determined over time. The results show thesurprising and dramatic reduction in mean tumor volume in mice for theangiostatin-expressing cells clones as compared with the non-transfectedand non-expressing control cells.

[0260] The mouse DNA sequence coding for mouse angiostatin protein isderived from mouse plasminogen cDNA. The mouse angiostatin encompassesmouse plasminogen kringle regions 1-4. The schematic for constructingthis clone is shown in FIG. 24.

[0261] The mouse angiostatin protein clones were transfected into T241fibrosarcoma cells using the LIPOFECTIN™ transfection system (availablefrom Life Technologies, Gaithersburg, Md.). The LIPOFECTIN™ reagent is a1:1 (w/w) liposome formulation of the cationic lipidN-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA),and diolecoyl phosphotidylethanolamine (DOPE) in membrane filteredwater.

[0262] The procedure for transient transfection of cells is as follows:

[0263] 1. T241 cells are grown in 60 cm² tissue culture dishes,seed≈1-2×10⁵ cells in 2 ml of the appropriate growth medium supplementedwith serum.

[0264] 2. Incubate the cells at 37° C. in a CO₂ incubator until thecells are 40-70% confluent. will usually take 18-24 h, but the time willvary among cell types. The T241 tumor cells confluency was approximately70%.

[0265] 3. Prepare the following solutions in 12×75 mm sterile tubes:

[0266] Solution A: For each transfection, dilute 5 μg of DNA in 100 μlof serum-free OPTI-MEM I Reduced Serum Medium (available from LifeTechnologies) (tissue culture grade deionized water can also be used).

[0267] Solution B: For each transfection, dilute 30 μg of LIPOFECTIN in100 μl OPTI-MEM medium.

[0268] 4. Combine the two solutions, mix gently, and incubate at roomtemperature for 10-15 min.

[0269] 5. Wash cells twice with serum-free medium.

[0270] 6. For each transfection, add 0.8 ml serum-free medium to eachtube containing the LIPOFECTIN™ reagent-DNA complexes. Mix gently andoverlay the complex onto cells.

[0271] 7. Incubate the cells for approximately 12 h at 37° C. in a CO₂incubator.

[0272] 8. Replace the DNA containing medium with 1 mg/ml selectionmedium containing serum and incubate cells at 37° C. in a CO₂ incubatorfor a total of 48-72 h.

[0273] 9. Assay cell extracts for gene activity at 48-72 h posttransfection.

[0274] Transfected cells can be assayed for expression of angiostatinprotein using angiostatin-specific antibodies. Alternatively, afterabout 10-14 days, G418 resistant colonies appeared in theCMV-angiostatin transfected T241 cells. Also, a number of clones wereseen in the vector alone transfected clones but not in the untransfectedclones. The G418 resistant clones were selected for their expression ofangiostatin, using a immunofluorence method.

[0275] Interestingly, the in vitro cell growth T241 cells and Lewis lungcells transfected with angiostatin was not inhibited or otherwiseadversely affected, as shown in FIGS. 25 and 26.

[0276]FIG. 27 depicts the results of the transfection experiment. Allthree of the angiostatin-expressing T241 transfected clones producedmean tumor volumes in mice that were substantially reduced relative tothe tumor volume in contol mice. The mean tumor volume of the miceimplanted with Clone 37 was only 13% of the control, while Clone 31 andClone 25 tumor volumes were only 21% and 34% of the control tumorvolumes, respectively. These results demonstrate that the DNA sequencescoding for angiostatin can be transfected into cells, that thetransfected DNA sequences are capable of expressing angiostatin proteinby implanted cells, and that the expressed angiostatin fucntions in vivoto reduce tumor growth.

EXAMPLE 24

[0277] Localization of in vivo Site of Angiostatin Expression

[0278] To localize the in vivo site of expression of angiostatinprotein, total RNA from various cell types, Lewis lung carcinoma cells(mouse), T241 fibrosarcoma (mouse), and Burkitt's lymphoma cells(human), both from fresh tumor or cell culture after several passageswere analysed to determine the presence of angiostatin transcripts.Northern analysis of samples showed an absence of any signal hybridizingwith thn sequence from all samples except that of normal mouse liver RNAshowing a single signal of approximately 2.4 kb corresponding to mouseplasminogen. Northern analysis of human samles show an absence of anysignal hybridizing with human angiostatin sequence from all samplesexcept that of normal human liver RNA showing a single signal ofapproximately 2.4 kb corresponding to human plasminogen.

[0279] Reverse transcription polymerase chain reaction (RT-PCR) analysisshowed an absence of any product from all samples probed with mouseangiostatin sequences except that of the normal mouse liver. RT-PCRanalysis showed an absence of any product from all human samples probedwith human angiostatin sequences except that of the normal human liver(expected size of 1050 bp for mouse and 1134 bp for human).

[0280] Thus it appears that mouse angiostatin transcripts (assumingidentity with amino acids 97 to 450 of mouse plasminogen) are notproduced by all the above mouse samples and human angiostatintranscripts (assuming identity with amino acids 93 to 470 of humanplasminogen) are not produced by the above human samples. The positivesignals obtained in normal mouse/human liver is from hybridization withplasminogen.

EXAMPLE 25

[0281] Expression of Angiostatin in Yeast

[0282] The gene fragment encoding amino acids 93 to 470 of humanplasminogen was cloned into the XhoI/EcoRI site of pHIL-SI(Invitrogen)which allows the secreted expression of proteins using the PHO1secretion signal in the yeast Pichia pastoris. Similarly, the genefragment encoding amino acids 93 to 470 of human plasminogen was clonedinto the SnaBI/EcoRI site of pPIC9 (Invitrogen) which allows thesecreted expression of proteins using the a-factor secretion signal inthe yeast Pichia pastoris. The expressed human angiostatin proteins inthese systems will have many advantages over those expressed in E. colisuch as protein processing, protein folding and posttranslationalmodification inclusive of glycosylation.

[0283] Expression of gene in P. pastoris: is described in) Sreekrishna,K. et al. (1988) High level expression of heterologous proteins inmethylotropic yeast Pichia pastoris. J. Basic Microbiol. 29 (4):265-278, and Clare, J. J. et al. (1991) Production of epidermal growthfactor in yeast: High-level secretion using Pichia pastoris strainscontaining multiple gene copies, Gene 105:205-212, both of which arehereby incorporated herein by reference.

EXAMPLE 26

[0284] Expression of Angiostatin Proteins in Transgenic Animals andPlants

[0285] Transgenic animals such as of the bovine or procine family arecreated which express the angiostatin gene transcript. The transgenicanimal express angiostatin protein for example in the milk of theseanimals. Additionally edible transgenic plants which express theangiostatin gene transcript are constructed.

[0286] Constructing transgenic animals that express foreign DNA isdescribed in Smith H. Phytochrome transgenics: functional, ecologicaland biotechnical applications, Semin. Cell. Biol. 1994 5(5):315-325,which is hereby incorporated herein by reference.

EXAMPLE 27

[0287] Characterization of Endothelial Cell Proliferation InhibitingAngiostatin Fragments

[0288] The following example characterizes the activity of individualand combinational angiostatin fragments. The data suggests that afunctional difference exists among individual kringle structures, andpotent anti-endothelial, and hence anti-angiogenic, activity can beobtained from such protein fragments of angiostatin.

[0289] As used herein, “angiostatin fragment” means a protein derivativeof angiostain, or plasminogen, having an endothelial cell proliferationinhibiting activity. Angiostatin fragments are useful for treatingangiogenic-mediated diseases or conditions. For example, angiostatinfragments can be used to inhibit or suppress tumor growth. The aminoacid sequence of such an angiostatin fragment, for example, can beselected from a portion of murine plasminogen (SEQ ID NO:1), murineangiostatin (SEQ ID NO:2); human angiostatin (SEQ ID NO:3), Rhesusangiostatin (SEQ ID NO:4), porcine angiostatin (SEQ ID NO:5), and bovineangiostatin (SEQ ID NO:6), unless indicated otherwise by the context inwhich it is used.

[0290] As used herein, “kringle 1” means a protein derivative ofplasminogen having an endothelial cell inhibiting activity oranti-angiogenic activity, and having an amino acid sequence comprising asequence homologous to kringle 1, exemplified by, but not limited tothat of murine kringle 1 (SEQ ID NO:7), human kringle 1 (SEQ ID NO:8),Rhesus kringle 1 (SEQ ID NO:9), porcine kringle 1 (SEQ ID NO:10), andbovine kringle 1 (SEQ ID NO:11), unless indicated otherwise by thecontext in which it is used. Murine kringle 1 (SEQ ID NO:7) correspondsto amino acid positions 103 to 181 (inclusive) of murine plasminogen ofSEQ ID NO:1, and corresponds to amino acid positions 6 to 84 (inclusive)of murine angiostatin of SEQ ID NO:2. Human kringle 1 (SEQ ID NO:8),Rhesus kringle 1 (SEQ ID NO:9), porcine kringle 1 (SEQ ID NO:10), andbovine kringle 1 (SEQ ID NO:11) correspond to amino acid positions 6 to84 (inclusive) of angiostatin of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,and SEQ ID NO:6, respectively.

[0291] As used herein, “kringle 2” means a protein derivative ofplasminogen having an endothelial cell inhibiting activity oranti-angiogenic activity, and having an amino acid sequence comprising asequence homologous to kringle 2, exemplified by, but not limited tothat of murine kringle 2 (SEQ ID NO:12), human kringle 2 (SEQ ID NO:13),Rhesus kringle 2 (SEQ ID NO:14), porcine kringle 2 (SEQ ID NO:15), andbovine kringle 2 (SEQ ID NO:16), unless indicated otherwise by thecontext in which it is used. Murine kringle 2 (SEQ ID NO:12) correspondsto amino acid positions 185 to 262 (inclusive) of murine plasminogen ofSEQ ID NO:1, and corresponds to amino acid positions 88 to 165(inclusive) of murine angiostatin of SEQ ID NO:2. Human kringle 2 (SEQID NO:13), Rhesus kringle 2 (SEQ ID NO:14), porcine kringle 2 (SEQ IDNO:15), and bovine kringle 2 (SEQ ID NO:16) correspond to amino acidpositions 88 to 165 (inclusive) of angiostatin of SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0292] As used herein, “kringle 3” means a protein derivative ofplasminogen having an endothelial cell inhibiting activity oranti-angiogenic activity, and having an amino acid sequence comprising asequence homologous to kringle 3, exemplified by, but not limited tothat of murine kringle 3 (SEQ ID NO:17), human kringle 3 (SEQ ID NO:18),Rhesus kringle 3 (SEQ ID NO:19), porcine kringle 3 (SEQ ID NO:20), andbovine kringle 3 (SEQ ID NO:21). Murine kringle 3 (SEQ ID NO:17)corresponds to amino acid positions 275 to 352 (inclusive) of murineplasminogen of SEQ ID NO:1, and corresponds to amino acid positions 178to 255 (inclusive) of murine angiostatin of SEQ ID NO:2. Human kringle 3(SEQ ID NO:18), Rhesus kringle 3 (SEQ ID NO:19), porcine kringle 3 (SEQID NO:20), and bovine kringle 3 (SEQ ID NO:21) correspond to amino acidpositions 178 to 255 (inclusive) of angiostatin of SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0293] As used herein, “kringle 4” means a protein derivative ofplasminogen having an endothelial cell inhibiting activity oranti-angiogenic activity, and having an amino acid sequence comprising asequence homologous to kringle 4, exemplified by, but not limited tothat of murine kringle 4 (SEQ ID NO:22) and human kringle 4 (SEQ IDNO:23), unless indicated otherwise by the context in which it is used.Murine kringle 4 (SEQ ID NO:22) corresponds to amino acid positions 377to 454 (inclusive) of murine plasminogen of SEQ ID NO:1.

[0294] As used herein, “kringle 2-3” means a protein derivative ofplasminogen having an endothelial cell inhibiting activity oranti-angiogenic activity, and having an amino acid sequence comprising asequence homologous to kringle 2-3, exemplified by, but not limited tothat of murine kringle 2-3 (SEQ ID NO:24), human kringle 2-3 (SEQ IDNO:25), Rhesus kringle 2-3 (SEQ ID NO:26), porcine kringle 2-3 (SEQ IDNO:27), and bovine kringle 2-3 (SEQ ID NO:28), unless indicatedotherwise by the context in which it is used. Murine kringle 2-3 (SEQ IDNO:24) corresponds to amino acid positions 185 to 352 (inclusive) ofmurine plasminogen of SEQ ID NO:1, and corresponds to amino acidpositions 88 to 255 (inclusive) of murine angiostatin of SEQ ID NO:2.Human kringle 2-3 (SEQ ID NO:25), Rhesus kringle 2-3 (SEQ ID NO:26),porcine kringle 2-3 (SEQ ID NO:27), and bovine kringle 2-3 (SEQ IDNO:28) correspond to amino acid positions 88 to 255 (inclusive) ofangiostatin of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6,respectively.

[0295] As used herein, “kringle 1-3” means a protein derivative ofplasminogen having an endothelial cell inhibiting activity oranti-angiogenic activity, and having an amino acid sequence comprising asequence homologous to kringle 1-3, exemplified by, but not limited tothat of murine kringle 1-3 (SEQ ID NO:29), human kringle 1 (SEQ IDNO:30), Rhesus kringle 1-3 (SEQ ID NO:31), porcine kringle 1-3 (SEQ IDNO:32), and bovine kringle 1-3 (SEQ ID NO:33), unless indicatedotherwise by the context in which it is used. Murine kringle 1-3 (SEQ IDNO:29) corresponds to amino acid positions 103 to 352 (inclusive) ofmurine plasminogen of SEQ ID NO:1, and corresponds to amino acidpositions 6 to 255 (inclusive) of murine angiostatin of SEQ ID NO:2.Human kringle 1-3 (SEQ ID NO:30), Rhesus kringle 1-3 (SEQ ID NO:31),porcine kringle 1-3 (SEQ ID NO:32), and bovine kringle 1-3 (SEQ IDNO:33) correspond to amino acid positions 6 to 255 (inclusive) ofangiostatin of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6,respectively.

[0296] As used herein, “kringle 1-2” means a protein derivative ofplasminogen having an endothelial cell inhibiting activity oranti-angiogenic activity, and having an amino acid sequence comprising asequence homologous to kringle 1-2, exemplified by, but not limited tothat of murine kringle 1-2 (SEQ ID NO:34), human kringle 1-2 (SEQ IDNO:35), Rhesus kringle 1-2 (SEQ ID NO:36), porcine kringle 1-2 (SEQ IDNO:37), and bovine kringle 1-2 (SEQ ID NO:38), unless indicatedotherwise by the context in which it is used. Murine kringle 1-2 (SEQ IDNO:34) corresponds to amino acid positions 103 to 262 (inclusive) ofmurine plasminogen of SEQ ID NO:1, and corresponds to amino acidpositions 6 to 165 (inclusive) of murine angiostatin of SEQ ID NO:2.Human kringle 1-2 (SEQ ID NO:35), Rhesus kringle 1-2 (SEQ ID NO:36),porcine kringle 1-2 (SEQ ID NO:37), and bovine kringle 1-2 (SEQ IDNO:38) correspond to amino acid positions 6 to 165 (inclusive) ofangiostatin of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6,respectively.

[0297] As used herein, “kringle 1-4” means a protein derivative ofplasminogen having an endothelial cell inhibiting activity oranti-angiogenic activity, and having an amino acid sequence comprising asequence homologous to kringle 1-4, exemplified by, but not limited tothat of murine kringle 1-4 (SEQ ID NO:39) and human kringle 1-4 (SEQ IDNO:40), unless indicated otherwise by the context in which it is used.Murine kringle 1-4 (SEQ ID NO:39) corresponds to amino acid positions103 to 454 (inclusive) of murine plasminogen of SEQ ID NO:1.

[0298] Kringle 1, kringle 2, kringle 3, kringle 4, kringle 2-3, kringle1-3, kringle 1-2 and kringle 1-4 amino acid sequences are respectivelyhomologous to the specific kringle sequences identified above.Preferably, the amino acid sequences have a degree of homology to thedisclosed sequences of at least 60%, more preferably at least 70%, andmore preferably at least 80%. It should be understood that a variety ofamino acid substitutions, additions, deletions or other modifications tothe above listed fragments may be made to improve or modify theendothelial cell proliferation inhibiting activity or anti-angiogenicactivity of the angiostatin fragments. Such modifications are notintended to exceed the scope and spirit of the claims. For example, toavoid homodimerization by formation of inter-kringle disulfide bridges,the cysteine residues C4 in recombinant human kringle 2 (SEQ ID NO:13)and C42 in recombinant kringle 3 (SEQ ID NO:18) were mutated to serines.Furthermore, it is understood that a variety of amino acidsubstitutions, additions, deletions or other modifications can be madein the above identified angiostatin fragments, which do notsignificantly alter the fragments' endothelial cell proliferationinhibiting activity, and which are, therefore, not intended to exceedthe scope of the claims. By “not significantly alter” is meant that theangiostatin fragment has at least 60%, more preferably at least 70%, andmore preferably at least 80% of the endothelial cell proliferationinhibiting activity compared to that of the closest homologousangiostatin fragment disclosed herein.

[0299] Gene Construction and Expression

[0300] A PCR-based method was used to generate the cDNA fragments codingfor kringle 1 (K1), kringle 2 (K2), kringle 3 (K3), kringle 4 (K4) andkringle 2-3 (K2-3) of human plasminogen (HPg). Recombinant kringle 1(rK1), kringle 2 (rK2), kringle 3 (rK3), kringle 4 (rK4) and kringle 2-3(rK2-3) were expressed in E. coli as previously described (Menhart, N.,Shel, L. C., Kelly, R. F., and Castellino, F. J. (1991) Biochem. 30,1948-1957; Marti, D., Schaller, J., Ochensberger, B., and Rickli, E. E.(1994) Eur. J. Biochem. 219, 455-462; Söhndel, S., Hu, C.-K., Marti, D.,Affolter, M., Schaller, J., Llinas, M., and Rickli, E. E. (1996)Biochem.. in press; Rejante, M. R., Byeon, I.-J. L., and Llinas, M.(1991) Biochem. 30, 11081-11092). To avoid homodimerization by formationof inter-kringle disulfide bridges as shown in FIG. 32B, the cysteineresidues C169 and rK2 and C297 in rK3 were mutated to serines as seen inSEQ ID NO.s 13 and 18, at positions 4 and 42, respectively. (Söhndel,S., Hu, C.-K., Marti, D., Affolter, M., Schaller, J., Llinas, M., andRickli, E. E. (1996) Biochem. in press). The rK3 and rK2-3 contained anN-terminal hexa-histidine tag which was used for protein purification(not shown).

[0301] Proteolytic Digestion

[0302] The fragments of K1-3, K1-4 and K4 were prepared by digestion ofLys-HPg (Abbott Labs) with porcine elastase (Sigma) as previouslydescribed (Powell, J. R., and Castellino, F. J. (1983) Biochem. 22,923-927). Briefly, 1.5 mg elastase was incubated at room temperaturewith 200 mg of human plasminogen in 50 mM Tris-HCl pH 8.0 overnight withshaking. The reaction was terminated by the addition of diisopropylfluorophosphate (DFP) (Sigma) to a final concentration of 1 mM. Themixture was rocked for an additional 30 minutes at room temperature anddialyzed overnight against 50 mM Tris-HCl, pH 8.0.

[0303] Protein Purification

[0304] Recombinant K1 was expressed in DH5α E. coli bacterial cellsusing a pSTII plasmid vector. This protein was purified to homogeneityby chromatography using lysine-Sepharose 4B (Pharmacia) and Mono Q(BioRad) columns. E. coli bacterial cells (strain HB101) expressing rK2and rK3 were grown to an OD₆₀₀ of approximately 0.8 at 3° C. in 2×YTmedium containing 100 mg/ml ampicillin and 25 mg/ml kanamycin. IPTG(isopropyl-b-D-thiogalactopyranoside) was added to a final concentrationof 1 mM and cells were grown for an additional 4.5 hours at 37° C. toinduce the production of recombinant proteins. The cells were harvestedby centrifugation and the pellets were stored at −80° C. The thawed celllysates were re-suspended in the extraction buffer (6 M guanidinehydrochloride in 0.1M sodium phosphate, pH 8.0). The suspension wascentrifuged at 15,000×g for 30 minutes and b-mercaptoethanol was addedto the supernatant at a final concentration of 10 mM. The supernatantwas then loaded on a Ni²⁺-NTA agarose column (1.5 cm×5 cm)pre-equilibrated with the extraction buffer. The column was washedsuccessively with extraction buffer at pH 8.0 and pH 6.3, respectively.Recombinant K2 and K3 were eluted with extraction buffer at pH 50.

[0305] The proteolytically cleaved fragments of K1-3, K1-4 and K4 werepurified using a lysine-Sepharose 4B column (2.5 cm×15 cm) equilibratedwith 50 mM Tris-HCl, pH 8.0 until an absorbance at 180 nm reached 0.005.The absorbed kringle fragments were eluted with Tris buffer containing200 mM ε-aminocaproic acid, pH 8.0. The eluted samples were the dialyzedovernight against 20 mM Tris-HCl, pH 5.0, and were applied, to a BioRadMono-S column equilibrated with the same buffer. The fragments of K4,K1-3 and K1-4 were eluted with 0-20%, 20-50% and 50-70% step-gradientsof 20 mM phosphate/1 M KCl, pH 5.0. Most K1-3 and K1-4 fragments wereeluted from the column with 0.5 M KCl as determined by SDS-PAGE. Allfractions were dialyzed overnight against 20 mM Tris-HCl, pH 8.0. Afterdialysis, K1-3 and K1-4 fragments were further purified using aheparin-Sepharose column (5 cm×10 cm) (Sigma) pre-equilibrated with 20mM Tris-HCl buffer, pH 8.0. The K1-3 fragment was eluted with 350 mM KCland K1-4 was recovered from the flow-through fraction. The purifiedkringle fragments were analyzed on SDS-gels follows by silver-staining,by Western immunoblotting analysis with anti-human K4 and K1-3polyclonal antibodies, and by amino-terminal sequencing analysis.

[0306] In vitro Re-Folding

[0307] The re-folding of rK2, rK3 and rK2-3 was performed according to astandard protocol (Cleary, S., Mulkerrin, M. G., and Kelley, R. R.(1989) Biochem. 28, 1884-1891). The purified proteins were adjusted topH 8.0 and dithiotreitol (DTT) was added to a final concentration of 5mM. After an overnight incubation, the solution was diluted with 4volumes of 50 mM Tris-HCl, pH 8.0, containing 1.25 mM reducedglutathione. After 1 hour of incubation, oxidized glutathione was addedto a final concentration of 1.25 mM and incubated for 6 hours at 4° C.The renatured protein was dialyzed initially against H₂O for 2 days andfor an additional two days against 50 mM phosphate-buffered saline, pH8.0. The solution was then loaded onto a lysine-Bio-Gel column (2 cm×13cm) equilibrated with the same phosphate-buffered saline. The column waswashed with phosphate-buffered saline and protein was eluted with aphosphate buffer containing 50 mM 6-AHA (6-aminohexanoic acid).Reverse-phase HPLC was performed on an Aquapore Butyl column (2.1×100mm, widepore 30 nm, 7 mm, Applied Biosystems) and a Hewlett Packardliquid chromatography was used with acetonitrile gradients.

[0308] Reduction and Alkylation

[0309] The reduction and alkylation of kringle fragments were performedaccording to a standard protocol (Cao, Y., and Pettersson, R. F., (1990)Growth Factors 3, 1013). Approximately 20-80 mg of purified proteins in300-500 ml DME medium in the absence of serum were incubated at roomtemperature with 15 ml of 0.5 M DTT for 15 minutes. After incubation, 30ml of 0.5 M iodoacetamide was added to the reaction. The proteinsolution was dialyzed at 4° C. overnight initially against 20 volumes ofDMEM. The solution was further dialyzed at 4° C. for an additional 4hours against 20 volumes of fresh DMEM. After dialysis, the samples wereanalyzed on a SDS-gel and assayed for their inhibitory activities onendothelial cell proliferation.

[0310] Endothelial Proliferation Assay

[0311] Bovine capillary endothelial (BCE) cells were isolated aspreviously described (Folkman, J., Haudenschild, C. C., and Zetter, B.R. (1979) Proc. Natl. Acad. Sci USA. 76, 5217-5121) and maintained inDMEM supplemented with 10% heat-inactivated bovine calf serum (BCS),antibiotics, and 3 ng/ml recombinant human bFGF (Scios Nova,Mountainview, Calif.). Monolayers of BCE cells growing in 6-well plateswere dispersed in a 0.05% trypsin solution. Cells were re-suspended withDMEM containing 10% BCS. Approximately 12,500 cells in 0.5 ml were addedto each well of gelatinized 24-well tissue culture plates and incubatedat 37° C. (in 10% CO₂) for 24 hours. The medium was replaced with 500 mlof fresh DMEM containing 5% BCS and samples of individual orcombinatorial kringle fragments in triplicates were added to each well.After 30 minutes of incubation, bFGF was added to a final concentrationof 1 ng/ml. After 72 hours of incubation, cells were trypsinized,re-suspended in Hematall (Fisher Scientific, Pittsburg, Pa.) and countedwith a Coulter counter.

[0312] Purification and Characterization of Kringle Fragment of HumanPlasminogen

[0313] The cDNA fragments coding for individual kringles (K1, K2, K3,and K4) and kringles 2-3 (K2-3) of human plasminogen were amplified by aPCR-based method (FIG. 28). The PCR-amplified cDNA fragments were clonedinto a bacterial expression vector. Recombinant proteins expressed fromEscherichia coli were refolded in vitro and were purified to >98%homogeneity using HPLC-coupled chromatography (FIG. 29). Under reducingconditions, recombinant K2, K3 and K4 migrated with molecular weights of12-13 kDa (FIG. 29A, lanes 2-4), corresponding to the predictedmolecular weights of each kringle fragment. Recombinant K1 migratingwith a higher molecular weight of 17 kDa was identified by SDS-gelelectrophoresis. The fragments of K1-4 and K1-3 were obtained byproteolytic digestion of human Lys-plasminogen (Lys-HPg) with elastaseas previously described (Powell, J. R., and Castellino, F. J. (1983)Biochem. 22, 923-927; Brockway, W. J., and Castellino, F. J. (1972)Arch. Biochem. Biophys). These two fragments (FIG. 29B, lanes 1 and 2)with predicted molecular weights of 43 kDa and 35 kDa, respectively,were also purified to homogeneity. N-terminal amino acid sequenceanalysis of the purified fragments yielded an identical sequence,-YLSE-, followed by SEQ ID NO:30 and SEQ ID NO:40, for K1-3 and K1-4,respectively. The N-terminal sequence for K4 produced -VVQD- withapproximately 20% -VQD-, followed by SEQ ID NO:23, each of which ispredicted from the expected sequence beginning with Valine¹⁷⁶ andValine¹⁷⁷ of human angiostatin (SEQ ID NO: 3).

[0314] Anti-Endothelial Cell Proliferative Activity of IndividualKringles

[0315] Individual recombinant kringle fragments of angiostatin wereassayed for the inhibitory activities on bovine capillary endothelial(BCE) cell growth stimulated by bFGF. As shown in FIG. 30A, rK1inhibited BCE cell proliferation in a dose-dependent fashion. Theconcentration of rK1 required to reach 50% inhibition (ED₅₀) was about320 nM (Table 4). In contrast, rK4 exhibited little or no inhibitoryeffect on endothelial cell proliferation. Recombinant K2 and rK3, twonon-lysine binding kringle fragments, also produced a dose-dependentinhibition of endothelial cell proliferation (FIG. 30B). However, theinhibitory potency of rK2 was substantially lower than rK1 and rK3(ED₅₀=460) (FIG. 30 and Table 4). No cytotoxicity or distinct morphologyassociated with apoptotic endothelial cells such as rounding,detachment, and fragmentation of cells could be detected, even afterincubation with a high concentration of these kringle fragments. Thesedata suggest that the anti-endothelial growth activity of angiostatinmay be shared by fragments of K1, K2 and K3, and lesser so by K4. TABLE4 Inhibitory activity on capillary endothelial cell proliferation.Fragments ED₅₀ (nM) Kringle 1 320 Kringle 2 — Kringle 3 460 Kringle 4 —Kringle 2-3 — Kringle 1-3  70 Kringle 1-4 (Angiostatin) 135

[0316] Anti-Endothelial Cell Proliferative Activity of K1-3 and K1-4Fragments

[0317] To evaluate the anti-endothelial cell proliferative effect ofcombined kringle fragments, purified proteolytic fragments of humanK1-4, K1-3 and rK2-3 were assayed on BCE cells. In agreement withprevious findings (O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C.,Rosenthal, R. A., Moses, J., Lane, W. S., Cao, Y., Sage, E. H., andFolkman, J. (1994) Cell 79, 315-328), BCE cell proliferation, as shownin FIG. 31, was significantly inhibited by angiostatin-like fragmentK1-4 (ED₅₀=135 nM) (Table 4). An increase of anti-endothelial growthactivity was obtained with K1-3 fragment (ED₅₀=70 nM) (Table 4). Theinhibition of endothelial cell proliferation occurred in adose-dependent manner. These results indicate that removal of K4 fromangiostatin potentiates anti-endothelial growth activity.

[0318] Additive Inhibition by rK2 and rK3

[0319] The fragment of rK2-3 displayed only weak inhibitory activitywhich was similar to that of rK2 alone (FIG. 31). However, both rK2 andrK3 inhibited endothelial cell proliferation (FIG. 30B). This findingsuggested that the inhibitory effect of K3 was hidden in the structureof K2-3. Previous structural studies showed that an inter-kringledisulfide bond was present between K2 (cysteine¹⁶⁹) and K3 (cysteine²⁹⁷)of human plasminogen, corresponding to cysteine⁹¹ and cysteine²¹⁹ of SEQID NO: 3 (Söhndel, S., Hu, C.-K., Marti, D., Affolter, M., Schaller, J.,Llinas, M., and Rickli, E. E. (1996) Biochem. in press) See FIG. 32B.The inhibitory effect of rK2 and rK3 in combination was tested.Interestingly, an additive inhibition was seen when individual rK2 andrK3 fragments were added together to BCE cells. See FIG. 32A. Theseresults imply that it is preferable to open the interdisulfide bridgebetween K2 and K3 in order to obtain the maximal inhibitory effect ofK2-3.

[0320] Appropriate Folding of Kringle Structures is Required for theAnti-Endothelial Activity of Angiostatin

[0321] To study whether the folding of kringle structures is requiredfor the anti-endothelial proliferation activity, native angiostatin wasreduced with DTT and assayed on bovine capillary endothelial cells.After reduction, angiostatin was further alkylated with iodoacetamideand analyzed by SDS gel electrophoresis. As shown in FIG. 34A, theDTT-treated protein migrated at a higher position with molecular weightof about 42 kDa (lane 2) as compared to the native angiostatin withmolecular weight of 33 kDa (lane 1), suggesting that angiostatin wascompletely reduced. The anti-proliferation activity of angiostatin waslargely abolished after reduction (FIG. 34B). From these results, weconclude that the correct folding of angiostatin through theintra-kringle disulfide bonds is preferable to maintain its potenteffect on inhibition of endothelial cell proliferation.

[0322] Amino acid sequence alignment of the kringle domains of humanplasminogen shows that K1, K2, K3 and K4 display identical grossarchitecture and remarkable sequence homology (56-82% identify) as seenin FIG. 35. Among these structures, the high-affinity lysine bindingkringle, K1, is the most potent inhibitory segment of endothelial cellproliferation. Of interest, the intermediate-affinity lysine bindingfragment, K4, lacks inhibitory activity. These data suggest that thelysine binding site of the kringle structures may not be directlyinvolved in the inhibitory activity. The amino acid conservation andfunctional divergence of these kringle structures provide an idealsystem to study the role mutations caused by DNA replication duringevolution. Similar divergent activities relative to the regulation ofangiogenesis exhibited by a group of structurally related proteins arealso found in the -C-X-C- chemokine and prolactin-growth hormonefamilies (Maione, T. E., Gray, G. S., Petro, A. J., Hunt, A. L., andDonner, S. I. (1990) Science 247, 77-79.; Koch, A. E., Polverini, P. J.,Kunkel, S. L., Harlow, L. A., DiPietro, L. A., Elner, V. M., Elner, S.J., and Strieter, R. M. (1992) Science 258, 1798-1801.; Cao, Y., Chen,C., Weatherbee, J. A., Tsang, M., and Folkman, J. (1995) J. Exp. Med.182, 2069-2077.; Strieter, R. M., Polverini, P. J., Arenberg, D. A., andKunkel, S. L. (1995) Shock 4, 155-160.; Jackson, D., Volpert, O. V.,Bouck, N., and Linzer, D. I. H. (1994) Science 266, 1581-1584).

[0323] Further sequence analysis reveals that K4 contains two positivelycharged lysine residues adjacent to cysteines 22 and 78 (FIG. 35). ¹Hnuclear magnetic resonance (NMR) analysis shows that these 4 lysines,together with lysine 57, form the core of a positively charged domain inK4 (Llinas M, unpublished data), whereas other kringle structures lacksuch a positively charged domain. Whether this lysine-enriched domaincontributes to the loss of inhibitory activity of kringle 4 of humanplasminogen remains to be studied. K4 was previously reported tostimulate proliferation of other cell types and to increase the releaseof intracellular calcium (Donate, L. E., Gherardi, E., Srinivasan, N.,Sowdhamini, R., Aporicio, S., and Blundell, T. L. (1994) Prot. Sci. 3,2378-2394). The fact that removal of K4 from angiostatin potentiates itsinhibitory activity on endothelial cells suggests that this structuremay prevent some of the inhibitory effect of K1-3.

[0324] The mechanism underlying how angiostatin and its related kringlefragments specifically inhibit endothelial cell growth remainsuncharacterized. It is not yet clear whether the inhibition is mediatedby a receptor that is specifically expressed in proliferatingendothelial cells, or if angiostatin is internalized by endothelialcells and subsequently inhibits cell proliferation. Alternatively,angiostatin may interact with an endothelial cell adhesion receptor suchas integrin a_(v)b₃, blocking integrin-mediated angiogenesis (Brooks, P.C., Montgomery, A. M., Rosenfeld, M., Reisfeld R. A., Hu, T. Klier, G.,and Cheresh, D. A. (1994) Cell 79, 1157-1164). Of interest, Friedlanderet. al. (Friedlander, M., Brooks, P. C., Shaffer, R. W., Kincaid, C. M.,Varner, J. A., and Cheresh, D. A. (1995) 270, 1502) reported recentlythat in vivo angiogenesis in cornea or chorioallantoic membrane models(induced by bFGF and by tumor necrosis factor) was a_(v)b₃ integrindependent. However, angiogenesis stimulated by VEGF, transforming growthfactor a, or phorbol esters was dependent on a_(v)b₅. Antibodies to theindividual integrins specifically blocked one of these pathways, and acyclic protein antagonist of both integrins blocked angiogenesis inducedby each cytokine (Friedlander, M., Brooks, P. C., Shaffer, R. W.,Kincaid, C. M., Varner, J. A., and Cheresh, D. A. (1995) 270, 1502).Because bFGF- and VEGF-induced angiogenesis are inhibited byangiostatin, it may block a common pathway for these integrin-mediatedangiogenesis.

[0325] An increasing number of endogenous angiogenesis inhibitors havebeen identified in the last few decades (Folkman, J. (1995) N. Engl. J.Med. 333, 1757-1763). Of the nine characterized endothelial cellsuppressors, several inhibitors are proteolytic fragments. For example,the 16 kDa N-terminal fragment of human prolactin inhibits endothelialcell proliferation and blocks angiogenesis in vivo (Clapp, C., Martial,J. A., Guzman, R. C., Rentierdelrue, F., and Weiner, R. I. (1993)Endorinology 133, 1292-1299). In a recent paper, D'Angelo et. al.reported that the antiangiogenic 16 kDa N-terminal fragment inhibitedthe activation of mitogen-activated protein kinase (MAPK) by VEGF andbFGF in capillary endothelial cells (D'Angelo, G., Struman, I., Martial,J., and Weiner, R. (1995) Proc. Natl. Acad. Sci. 92, 6374-6378). Similarto angiostatin, the intact parental molecule of prolactin does notinhibit endothelial cell proliferation nor is it an angiogenesisinhibitor. Platelet factor 4 (PF-4) inhibits angiogenesis at highconcentrations (Maione, T. E., Gray, G. S., Petro, A. J., Hunt, A. L.,and Donner, S. I. (1990) Science 247, 77-79; Cao, Y., Chen, C.,Weatherbee, J. A., Tsang, M., and Folkman, J. (1995) J. Exp. Med. 182,2069-2077). However, the N-terminally truncated proteolytically cleavedPF-4 fragment exhibits a 30- to 50-fold increase in itsanti-proliferative activity over the intact PF-4 molecule (Gupta, S. K.,Hassel, T., and Singh, J. P. (1995) Proc. Natl. Acad. Sci. 92,7799-7803). Smaller protein fragments of fibronectin, murine epidermalgrowth factor, and thrombospondin have also been shown to specificallyinhibit endothelial cell growth (Homandberg, G. A., Williams, J. E.,Grant, D., Schumacher, B., and Eisenstein, R. (1985) Am. J. Pathol. 120,327-332; Nelson, J., Allen, W. E., Scott, W. N., Bailie, J. R., Walker,B., McFerran, N. V., and Wilson, D. J. (1995) Cancer Res. 55, 3772-3776;Tolsma, S. S., Volpert, O. V., Good, D. J., Frazer, W. A., Polverini, P.J., and Bouck, N. (1993) J. Cell Biol. 122, 497-511). Proteolyticprocessing of a large protein may change the conformational structure ofthe original molecule or expose new epitopes that are antiangiogenic.Thus, protease(s) may play a critical role in the regulation ofangiogenesis. To date, little is known about the regulation of theseprotease activities in vivo.

[0326] The data also show that the disulfide bond mediated folding ofthe kringle structures in angiostatin is preferable to maintain itsinhibitory activity on endothelial cell growth. Kringle structuresanalogous to those in plasminogen are also found in a variety of otherproteins. For example, apolipoprotein (a) has as many as 37 repeats ofplasminogen kringle 4 (McLean, J. W., Tomlinson, J. E., Kuang, W.-J.,Eaton, D. L., Chen, E. Y., Fless, G. M., Scanu, A. M., and Lawn, R. M.(1987) Nature 330, 132-137). The amino terminal portion of prothrombinalso contains two kringles that are homologous to those of plasminogen(Walz, D. A., Hewett-Emmett, D., and Seegers, W. H. (1977) Proc. Natl.Acad. Sci. 74, 1969-1973). Urokinase has been shown to possess a kringlestructure that shares extensive homology with plasminogen (Gunzler, W.A., J., S. G., Otting, F., Kim, S.-M. A., Frankus, E., and Flohe, L.(1982) Hoppe-Seyler's A. Physiol. Chem. 363, 1155-1165). In addition,surfactant protein B and hepatocyte growth factor (HGF), also carrykringle structures (Johansson, J., Curstedt, T., and Jörnvall., H.(1991) Biochem. 30, 6917-6921; Lukker, N. A., Presta, L. G., andGodowski, P. J. (1994) Prot. Engin. 7, 895-903).

EXAMPLE 28

[0327] Suppression of Metastases and of Endothelial Cell Proliferationby Angiostatin Fragments

[0328] The following example characterizes the activity of additionalangiostatin fragments. The data suggests that potent anti-endothelialand tumor suppressive activity can be obtained from such proteinfragments of angiostatin.

[0329] As used herein, “kringle 1-4BKLS” means a protein derivative ofplasminogen having an endothelial cell inhibiting activity, and havingan amino acid sequence comprising a sequence homologous to kringle1-4BKLS, exemplified by, but not limited to that of murine kringle1-4BKLS (SEQ ID NO:41), and human kringle 1-4BKLS (SEQ ID NO:42), unlessindicated otherwise by the context in which it is used. Murine kringle1-4BKLS (SEQ ID NO:41) corresponds to amino acid positions 93 to 470(inclusive) of murine plasminogen of SEQ ID NO:1. This exampledemonstrates that an “angiostatin fragment” can be a plasminogenfragment and encompass an amino acid sequence larger than theangiostatin presented in SEQ ID NO:3, for example, and still havetherapeutic endothelial cell proliferation inhibiting activity oranti-angiogenic activity.

[0330] A kringle 1-4BLKS amino acid sequence is homologous to thespecific kringle 1-4BLKS sequences identified above. Preferably, theamino acid sequences have a degree of homology to the disclosedsequences of at least 60%, more preferably at least 70%, and morepreferably at least 80%. It should be understood that a variety of aminoacid substitutions, deletions and other modifications to the abovelisted fragments may be made to improve or modify the endothelial cellinhibiting activity of the fragments. Such modifications are notintended to exceed the scope and spirit of the claims. Furthermore, itis understood that a variety of silent amino acid substitutions,additions, or deletions can be made in the above identified kringlefragments, which do not significantly alter the fragments' endothelialcell inhibiting activity, and which are, therefore, not intended toexceed the scope of the claims.

[0331] Cloning of Angiostatin in Pichia pastoris

[0332] Sequences encoding angiostatin were amplified by PCR using Ventpolymerase (New England Biolabs) and primers #154(5′-ATCGCTCGAGCGTTATTTGAAAAGAAAGTG-3′) (SEQ ID NO:43) and #151(5′-ATCGGAATTCAAGCAGGACAACAGGCGG-3′) (SEQ ID NO:44) containing linkersXhol and Eco R1 respectively and using the plasmid pTrcHis/HAs astemplate. This plasmid contained sequences encoding amino acids 93 to470 of human plasminogen (SEQ ID NO:42) for cloning into the Xho I/ECoR1 site of pHIL-S1 expression vector using the P. pastoris nativesecretion signal PHO 1. This same sequence was amplified in the samemanner using primers #156 (5′-ATCGTACGTATTATTTGAAAAGAAAGTG-3′) (SEQ IDNO:45) and #151 containing linkers Sna B1 and Eco RI respectively, forcloning into the Sna B1/ECo R1 site of expression vector pP1C9 with thealpha-factor secretory signal. The products of the amplifications weregel purified, linkers were digested with the appropriate enzymes, andagain purified using gene-clean (Bio 101). These gene fragments wereligated into the appropriate vectors. Resultant clones were selected andplasmid preparations of clones were obtained and linearized to generateHis⁺ Mut^(s) and His⁺ Mut⁺ recombinant strains when transformed into P.pastoris host strain GS115. Integration was confirmed by PCR.

[0333] Both His⁺ and His⁺ Mut⁺ recombinants were induced with methanoland screened for high expression of angiostatin using Coomassie stainedSDS-PAGE gels and immunoblots using mouse monoclonal antibody againstkringles 1 to 3 (Castellino, Enzyme Research Laboratories, Inc., SouthBend, Ind.). From these, a GS115 transformed P. pastoris clonepHIL-S1/HAs18 was selected and phenotypically characterized as His⁺Mut^(s).

[0334] Expression of PHIL-S1/HAs18

[0335] Expression of angiostatin from pHIL-S1/HAs18 was typical for aHis⁺ Mut^(s) clone. At induction in baffled shake flasks, 1 L of OD₆₀₀cells were cultured in 150 ml of buffered metanol complex mediumcontaining 1% yeast extract, 2% peptone, 100 mM potassium phosphate pH6.0, 1.34% yeast nitrogen base with ammonium sulfate, 0.00004% biotinand 0.5% methanol, in a 1 L baffled flask. Cells were constantly shakenat 30° C., 250 rpm. Methanol was batch fed at 24 hour intervals byaddition of absolute methanol to a final of 0.5%. After 120 hours cellswere spun at 5,000 rpm for 10 minutes, and supernatants were stored at−70° C. until used.

[0336] Purification of Angiostatin from P. pastoris Fermentation Brothby Lysine-Sepharose Chromatography

[0337] All procedures are carried out at 4° C. Crude fermentation broth,typically 200 ml, containing angiostatin was clarified by centrifugationat 14,000×g and concentrated by Centriprep 30 (amicon) 30 kDa molecularweight cutoff membrane to approximately one-fourth the original volume.One volume of 50 mM phosphate buffer, pH 7.5, was added to theconcentrated sample which was again concentrated by Centriprep toone-fourth the original sample volume. The sample was again dilutedvolume:volume with 50 mM sodium phosphate buffer, pH 7.5. 60 glysine-sepharose 4B (Pharmacia) was resuspended in 500 ml ice-cold 50 mMphosphate buffer, pH 7.5 and used to pack a 48×100 mm column (˜180 mlpacked volume). The column was washed overnight with 7.5 column volumes(CV) of 50 mM sodium phosphate buffer, pH 7.5, at a flow rate of 1.5ml/min. The sample was pumped onto the column at a flow rate of 1.5ml/min and the column washed with 1.5 CV of 50 mM sodium phosphate, pH7.5, at a flow rate of 3 ml/min. The column was then washed with 1.5 CVphosphate-buffered saline, pH 7.4, at a flow rate of 3 ml/min:angiostatin was then eluted with 0.2 M ε-amino-n-caproic acid, pH 7.4 ata flow rate of 3 ml/min. Fractions containing significant absorbancewere pooled and dialyzed for 24-48 hours against deionized water andlyophilized. A typical recovery from a 100 mg total protein load is 10mg angiostatin. Columns were regenerated using 5 column volumes of 50 mMsodium phosphate/1 M NaCl, pH 7.5.

[0338] Bovine Capillary Endothelial Cell Proliferation Assay

[0339] Bovine capillary endothelial cells were obtained as previouslydescribed. The cells are maintained in DMEM containing 3 mg/ml ofrecombinant human bFGF (Scios Nova, Mountainview, Calif.), supplementedwith 10% heat-inactivated bovine calf serum, 100 U/ml penicillin, 100mg/ml streptomycin, and 0.25 mg/ml fungizone (BioWhittaker) in 75 cm²cell-culture flasks. The assay was performed as described previously.

[0340] Animal Studies

[0341] Six to eight week old male C57BI/6J mice (Jackson Laboratories)were inoculated subcutaneously with murine Lewis lung carcinoma-lowmetastatic (LLC-LM) line (1×10⁶ cells/injection). Approximately 14 daysafter implantation, when primary tumor reached 1.5 cm³, animals wereanaesthetized with methoxyflurane and primary tumors were surgicallyexcised. The incision site was closed with simple interrupted sutures.Half the animals in this group received a loading dose (3 mg/kg by thesubcutaneous route) of recombinant or plasminogen derived angiostatinsubcutaneously immediately after surgery, followed by daily inoculationsof 1.5 mg/kg for 14 days. A control group of mice received an equalvolume of PBS every day for 14 days following surgery. All mice weresacrificed 14 days after primary tumor removal (28 days after tumorimplantation), lungs were removed and weighed, and surface metastaseswere counted with stereomicroscope.

[0342] Characteristics of Recombinant Human Angiostatin Fragments

[0343] A gene fragment encoding human angiostatin including kringles 1to 4 of human plasminogen that contains a total of 26 cysteines, wasexpressed in Pichia pastoris, the methylotropic yeast. P. pastorisexpressed angiostatin binds lysine sepharose and can be specificallyeluted by ε-amino caproic acid. This demonstrates that fully functionalepsilon amino caproic acid-binding kringle(s), which are physicalproperties of kringle 1 and 4 of plasminogen (Sottrup-Jensen, L. et al.,Progress in Chemical Fibrinolysis and Thrombolysis, Vol. 3 (1978) RavensPress, N.Y. p. 191), can be expressed and secreted by P. pastoris andpurified by techniques that do not require refolding (FIGS. 36A and B).Expressed angiostatin from P. pastoris as well as angiostatin purifiedby elastase cleavage of plasminogen were recognized by aconformationally dependent monoclonal antibody against kringle 1 to 3(Castellino, Enzyme Research Laboratories, Inc., South Bend, Ind.) (FIG.36B). This antibody fails to recognize reduced forms of plasminogen orangiostatin.

[0344]P. pastoris expressed angiostatin is seen as a doublet thatmigrates at 49 kDa and 51.5 kDa on denatured unreduced SDS-PAGECoomassie stained gels. P. pastoris expressed proteins arepost-translationally modified with the majority of N-linkedglycosylation of the high-mannose type and insignificant O-linkedglycosylation. To evaluate the possibility of glycosylation in P.pastoris expressed angiostatin, we digested the recombinant angiostatinwith endoglycosidase H specific for high mannose structures, causing the51.5 kDa band to migrate identically with the band at 49 kDa (FIGS. 37Aand B). O-glycanase digestion with prior neuraminidase treatment toremove sialic acid residues, did not change the pattern of migration ofthe doublet (data not shown). These results indicate that P. pastorisexpressed angiostatin in two forms: (1) with an N-linked complex chainprobably of the structure:

[0345] and (2) without any glycosylation.

[0346] Inhibition of Bovine Capillary Endothelial Cells In Vitro

[0347] To determine if recombinantly expressed angiostatin had thepotential for antiangiogenic activity, BCEs were cultured in thepresence of bFGF to determine if the addition of purified recombinantangiostatin would inhibit the proliferation of BCEs. Purified P.pastoris-expressed angiostatin inhibited the bFGF-driven proliferationof bovine endothelial cells in vitro (FIG. 38B) in a dose dependentmanner (FIG. 38C). At 1 ug/ml of recombinant angiostatin, inhibition was80%. The 50% inhibition was equivalent to that obtained with angiostatinderived from elastase cleavage of human plasminogen.

[0348] Suppression of Metastases In Vivo

[0349] The transplantable murine LLC (LM) line from which angiostatinwas first identified was used. When implanted subcutaneously in syngenicC57B1/6J mice, these tumors grow rapidly, producing >1.5 cm³ tumorswithin 14 days. Following primary tumor resection, the micometastases inthe lungs grow exponentially, to completely cover the surface of thelung. These metastases are highly vascularized by day 14 after primarytumor resection. If the primary tumor is left on, the micrometastasesremain dormant and are not macroscopically visible. Recombinantangiostatin was administered systemically to mice following primarytumor resection to test the suppression of the growth of metastases. P.pastoris expressed angiostatin administered systemically at 30ug/mouse/day inhibited the growth of metastases as quantitated byscoring of surface metastases (FIG. 39A) and total lung weight (FIG.39B). The weights of lungs of mice that had primary tumors resected andthat received daily doses of recombinant angiostatin or angiostatinobtained from elastase cleavage of plasminogen were of comparable tothose of normal mice (190 to 200 mg). Lungs of mice that had theirprimary tumors resected and subsequently treated with daily doses ofrecombinant angiostatin were pink with minimal numbers of unvascularizedmicrometastases (FIG. 40). In contrast, the mice treated with salineafter primary tumor resection had lungs covered with vascularizedmetastases (FIG. 41). Also of notable importance was an absence ofsystemic or local toxicity caused by P. pastoris expressed angiostatinat the dosage and regimen used in this study. There was no evidence ofinflammation or bleeding in all treated mice.

[0350] Angiostatin protein expressed by P. pastoris possesses twoimportant physical characteristics of the natural protein: (1) it isrecognized by a conformationally dependent monoclonal antibody raisedagainst kringle 1 to 3 of human plasminogen (FIG. 36B) and (2) it bindslysine (FIGS. 36A and B). These properties indicated that therecombinant angiostatin protein was expressed with a conformation thatmimics the native molecule. P. pastoris expressed angiostatin proteininhibits the proliferation of bovine capillary endothelial cellsstimulated by bFGF in vitro (FIG. 38). when administered systemically,the recombinant angiostatin maintained the otherwise lethal metastasticLewis lung carcinoma in a suppressed state (FIGS. 39A and B and FIG.40).

[0351] Preliminary data shows the absence of a detectable transcript forangiostatin in Lewis lung tumors freshly resected from mice or in LLCcells after 4 passages in in vitro culture. Plasminogen, produced by theliver, is maintained in circulation at a stable plasma concentration of1.6±0.2 μM. It is possible that LLC-LM tumors produce an enzyme thatcleaves plasminogen, bound or in circulation, to produce angiostatin.Alternatively inflammatory cells attracted to the tumor site couldproduce such an enzyme.

[0352] It is intriguing that both P. pastoris as well as native humanplasminogen is produced in a glycosylated and a non-glycosylated form.In the case of human plasminogen, a single transcript for a single genecan produce both forms. The molecular mechanism of differentialpost-translational modifications of human plasminogen, as well as thatseen in TPA are unknown.

[0353] Angiostatin is highly expressed by P. pastoris. Supernatantscontain 100 mg/L of the protein. Therefore, the quantities required forclinical trials should be straightforward to produce and purify usingstandard technology well-known to those skilled in the art. Thedevelopment of this expression system, and the demonstration of the invitro and in vivo activity of purified recombinant angiostatin againstmetastases provided the foundation for assessment of the capacity ofthese fragments to inhibit tumor growth and prolong life in cancerpatients and others suffering from angiogenic-mediated disease.

EXAMPLE 29

[0354] Kringle 1-5 Angiostatin Protein Fragment

[0355] The following example describes one method for the production ofkringle 1-5 angiostatin protein fragment.

[0356] As used herein, “kringle 1-5” means a protein derivative ofplasminogen having an endothelial cell inhibiting activity oranti-angiogenic activity, and having an amino acid sequence comprising asequence homologous to kringle 1-5, exemplified by, but not limited tothat of murine kringle 1-5 corresponding to amino acid positions 102 to560 (inclusive) of murine plasminogen of SEQ ID NO:1. Kringle 5 itselfis represented in the murine sequence of plasminogen of SEQ ID NO:1 atamino acid positions 481-560 (inclusive). The amino acid andcorresponding nucleotide sequence of plasminogen is provided in Forsgrenet al., “Molecular cloning and characterization of a full-length cDNAclone for human plasminogen,” FEBS 213:2, pp.254-260 (1987), which ishereby incorporated by reference.

[0357] Kringle 1-5 amino acid sequences are respectively homologous tothe specific kringle 1-5 sequence identified above. Preferably, theamino acid sequences have a degree of homology to the disclosedsequences of at least 60%, more preferably at least 70%, and morepreferably at least 80%. It should be understood that a variety of aminoacid substitutions, additions, deletions or other modifications to theabove listed fragments may be made to improve or modify the endothelialcell proliferation inhibiting activity or anti-angiogenic activity ofthe angiostatin fragments. Such modifications are not intended to exceedthe scope and spirit of the claims. For example, to avoidhomodimerization by formation of inter-kringle disulfide bridges, thecysteine residues can be mutated to serines. Furthermore, it isunderstood that a variety of amino acid substitutions, additions,deletions or other modifications can be made in the above identifiedangiostatin fragments, which do not significantly alter the fragments'endothelial cell proliferation inhibiting activity, and which are,therefore, not intended to exceed the scope of the claims. By “notsignificantly alter” is meant that the angiostatin fragment has at least60%, more preferably at least 70%, and more preferably at least 80% ofthe endothelial cell proliferation inhibiting activity compared to thatof the closest homologous angiostatin fragment disclosed herein.

[0358] Kringle 1-5 angiostatin protein fragment can be producedaccording to the following method:

[0359] 1) Convert purified human palsminogen (Plg) to Lys Plg using theenzyme plasmin.

[0360] 2) Digest Lys Plg with TPA or urokinase. This will result in theheavy (A) and light (B) chains, but still linked together by 2 disulfidebonds.

[0361] 3) These bonds can be specifically reduced by common reducingagents like beta mercaptoethanol to result in separate heavy chain A andlight chain B.

[0362] 4) Then block the cysteines so that they do not form bonds againby making the A and B chains into S-carboxymethyl derivatives (Robbins,K C, Bernabe P, Arzadon L, Summaria L. J. Biol. Chem. 247(21):6757-6762(1972). “The primary structure of human plasminogen. I. The NH2-terminalsequences of human plasminogen and the s-carboxymethyl heavy (A) andlight (B) chain derivatives of plasmin.”)

[0363] 5) Run the product of step 4 over a lysine-Sepharose column topurify K1-5 from the rest.

[0364] QED.

[0365] The kringle 1-5 angiostatin protein fragment can be used toinhibit endothelial cell proliferation and angiogenesis in vitro and invivo. In particular, the kringle 1-5 angiostatin protein fragment can beused to inhibit angiogenesis in a cancerous tumor.

[0366] It should be understood that the foregoing relates only topreferred embodiments of the present invention, and that numerousmodifications or alterations may be made therein without departing fromthe spirit and the scope of the invention as set forth in the appendedclaims.

1 52 1 812 PRT Murinae gen. sp. misc_feature Plasminogen 1 Met Asp HisLys Glu Val Ile Leu Leu Phe Leu Leu Leu Leu Lys Pro 1 5 10 15 Gly GlnGly Asp Ser Leu Asp Gly Tyr Ile Ser Thr Gln Gly Ala Ser 20 25 30 Leu PheSer Leu Thr Lys Lys Gln Leu Ala Ala Gly Gly Val Ser Asp 35 40 45 Cys LeuAla Lys Cys Glu Gly Glu Thr Asp Phe Val Cys Arg Ser Phe 50 55 60 Gln TyrHis Ser Lys Glu Gln Gln Cys Val Ile Met Ala Glu Asn Ser 65 70 75 80 LysThr Ser Ser Ile Ile Arg Met Arg Asp Val Ile Leu Phe Glu Lys 85 90 95 ArgVal Tyr Leu Ser Glu Cys Lys Thr Gly Ile Gly Asn Gly Tyr Arg 100 105 110Gly Thr Met Ser Arg Thr Lys Ser Gly Val Ala Cys Gln Lys Trp Gly 115 120125 Ala Thr Phe Pro His Val Pro Asn Tyr Ser Pro Ser Thr His Pro Asn 130135 140 Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Gln145 150 155 160 Gly Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg Tyr AspTyr Cys 165 170 175 Asn Ile Pro Glu Cys Glu Glu Glu Cys Met Tyr Cys SerGly Glu Lys 180 185 190 Tyr Glu Gly Lys Ile Ser Lys Thr Met Ser Gly LeuAsp Cys Gln Ala 195 200 205 Trp Asp Ser Gln Ser Pro His Ala His Gly TyrIle Pro Ala Lys Phe 210 215 220 Pro Ser Lys Asn Leu Lys Met Asn Tyr CysHis Asn Pro Asp Gly Glu 225 230 235 240 Pro Arg Pro Trp Cys Phe Thr ThrAsp Pro Thr Lys Arg Trp Glu Tyr 245 250 255 Cys Asp Ile Pro Arg Cys ThrThr Pro Pro Pro Pro Pro Ser Pro Thr 260 265 270 Tyr Gln Cys Leu Lys GlyArg Gly Glu Asn Tyr Arg Gly Thr Val Ser 275 280 285 Val Thr Val Ser GlyLys Thr Cys Gln Arg Trp Ser Glu Gln Thr Pro 290 295 300 His Arg His AsnArg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu 305 310 315 320 Glu AsnTyr Cys Arg Asn Pro Asp Gly Glu Thr Ala Pro Trp Cys Tyr 325 330 335 ThrThr Asp Ser Gln Leu Arg Trp Glu Tyr Cys Glu Ile Pro Ser Cys 340 345 350Glu Ser Ser Ala Ser Pro Asp Gln Ser Asp Ser Ser Val Pro Pro Glu 355 360365 Glu Gln Thr Pro Val Val Gln Glu Cys Tyr Gln Ser Asp Gly Gln Ser 370375 380 Tyr Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly Lys Lys Cys Gln Ser385 390 395 400 Trp Ala Ala Met Phe Pro His Arg His Ser Lys Thr Pro GluAsn Phe 405 410 415 Pro Asp Ala Gly Leu Glu Met Asn Tyr Cys Arg Asn ProAsp Gly Asp 420 425 430 Lys Gly Pro Trp Cys Tyr Thr Thr Asp Pro Ser ValArg Trp Glu Tyr 435 440 445 Cys Asn Leu Lys Arg Cys Ser Glu Thr Gly GlySer Val Val Glu Leu 450 455 460 Pro Thr Val Ser Gln Glu Pro Ser Gly ProSer Asp Ser Glu Thr Asp 465 470 475 480 Cys Met Tyr Gly Asn Gly Lys AspTyr Arg Gly Lys Thr Ala Val Thr 485 490 495 Ala Ala Gly Thr Pro Cys GlnGly Trp Ala Ala Gln Glu Pro His Arg 500 505 510 His Ser Ile Phe Thr ProGln Thr Asn Pro Arg Ala Asp Leu Glu Lys 515 520 525 Asn Tyr Cys Arg AsnPro Asp Gly Asp Val Asn Gly Pro Trp Cys Tyr 530 535 540 Thr Thr Asn ProArg Lys Leu Tyr Asp Tyr Cys Asp Ile Pro Leu Cys 545 550 555 560 Ala SerAla Ser Ser Phe Glu Cys Gly Lys Pro Gln Val Glu Pro Lys 565 570 575 LysCys Pro Gly Arg Val Val Gly Gly Cys Val Ala Asn Pro His Ser 580 585 590Trp Pro Trp Gln Ile Ser Leu Arg Thr Arg Phe Thr Gly Gln His Phe 595 600605 Cys Gly Gly Thr Leu Ile Ala Pro Glu Trp Val Leu Thr Ala Ala His 610615 620 Cys Leu Glu Lys Ser Ser Arg Pro Glu Phe Tyr Lys Val Ile Leu Gly625 630 635 640 Ala His Glu Glu Tyr Ile Arg Gly Leu Asp Val Gln Glu IleSer Val 645 650 655 Ala Lys Leu Ile Leu Glu Pro Asn Asn Arg Asp Ile AlaLeu Leu Lys 660 665 670 Leu Ser Arg Pro Ala Thr Ile Thr Asp Lys Val IlePro Ala Cys Leu 675 680 685 Pro Ser Pro Asn Tyr Met Val Ala Asp Arg ThrIle Cys Tyr Ile Thr 690 695 700 Gly Trp Gly Glu Thr Gln Gly Thr Phe GlyAla Gly Arg Leu Lys Glu 705 710 715 720 Ala Gln Leu Pro Val Ile Glu AsnLys Val Cys Asn Arg Val Glu Tyr 725 730 735 Leu Asn Asn Arg Val Lys SerThr Glu Leu Cys Ala Gly Gln Leu Ala 740 745 750 Gly Gly Val Asp Ser CysGln Gly Asp Ser Gly Gly Pro Leu Val Cys 755 760 765 Phe Glu Lys Asp LysTyr Ile Leu Gln Gly Val Thr Ser Trp Gly Leu 770 775 780 Gly Cys Ala ArgPro Asn Lys Pro Gly Val Tyr Val Arg Val Ser Arg 785 790 795 800 Phe ValAsp Trp Ile Glu Arg Glu Met Arg Asn Asn 805 810 2 339 PRT Murinae gen.sp. 2 Val Tyr Leu Ser Glu Cys Lys Thr Gly Ile Gly Asn Gly Tyr Arg Gly 15 10 15 Thr Met Ser Arg Thr Lys Ser Gly Val Ala Cys Gln Lys Trp Gly Ala20 25 30 Thr Phe Pro His Val Pro Asn Tyr Ser Pro Ser Thr His Pro Asn Glu35 40 45 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Gln Gly50 55 60 Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asn65 70 75 80 Ile Pro Glu Cys Glu Glu Glu Cys Met Tyr Cys Ser Gly Glu LysTyr 85 90 95 Glu Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Asp Cys Gln AlaTrp 100 105 110 Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ala LysPhe Pro 115 120 125 Ser Lys Asn Leu Lys Met Asn Tyr Cys His Asn Pro AspGly Glu Pro 130 135 140 Arg Pro Trp Cys Phe Thr Thr Asp Pro Thr Lys ArgTrp Glu Tyr Cys 145 150 155 160 Asp Ile Pro Arg Cys Thr Thr Pro Pro ProPro Pro Ser Pro Thr Tyr 165 170 175 Gln Cys Leu Lys Gly Arg Gly Glu AsnTyr Arg Gly Thr Val Ser Val 180 185 190 Thr Val Ser Gly Lys Thr Cys GlnArg Trp Ser Glu Gln Thr Pro His 195 200 205 Arg His Asn Arg Thr Pro GluAsn Phe Pro Cys Lys Asn Leu Glu Glu 210 215 220 Asn Tyr Cys Arg Asn ProAsp Gly Glu Thr Ala Pro Trp Cys Tyr Thr 225 230 235 240 Thr Asp Ser GlnLeu Arg Trp Glu Tyr Cys Glu Ile Pro Ser Cys Glu 245 250 255 Ser Ser AlaSer Pro Asp Gln Ser Asp Ser Ser Val Pro Pro Glu Glu 260 265 270 Gln ThrPro Val Val Gln Glu Cys Tyr Gln Ser Asp Gly Gln Ser Tyr 275 280 285 ArgGly Thr Ser Ser Thr Thr Ile Thr Gly Lys Lys Cys Gln Ser Trp 290 295 300Ala Ala Met Phe Pro His Arg His Ser Lys Thr Pro Glu Asn Phe Pro 305 310315 320 Asp Ala Gly Leu Glu Met Asn Tyr Cys Arg Asn Pro Asp Gly Asp Lys325 330 335 Gly Pro Trp 3 339 PRT Homo sapiens 3 Val Tyr Leu Ser Glu CysLys Thr Gly Asn Gly Lys Asn Tyr Arg Gly 1 5 10 15 Thr Met Ser Lys ThrLys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser 20 25 30 Thr Ser Pro His ArgPro Arg Phe Ser Pro Ala Thr His Pro Ser Glu 35 40 45 Gly Leu Glu Glu AsnTyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly 50 55 60 Pro Trp Cys Tyr ThrThr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp 65 70 75 80 Ile Leu Glu CysGlu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr 85 90 95 Asp Gly Lys IleSer Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp 100 105 110 Asp Ser GlnSer Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro 115 120 125 Asn LysAsn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu 130 135 140 ArgPro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys 145 150 155160 Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr 165170 175 Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val180 185 190 Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr ProHis 195 200 205 Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn LeuAsp Glu 210 215 220 Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro TrpCys His Thr 225 230 235 240 Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys LysIle Pro Ser Cys Asp 245 250 255 Ser Ser Pro Val Ser Thr Glu Gln Leu AlaPro Thr Ala Pro Pro Glu 260 265 270 Leu Thr Pro Val Val Gln Asp Cys TyrHis Gly Asp Gly Gln Ser Tyr 275 280 285 Arg Gly Thr Ser Ser Thr Thr ThrThr Gly Lys Lys Cys Gln Ser Trp 290 295 300 Ser Ser Met Thr Pro His ArgHis Gln Lys Thr Pro Glu Asn Tyr Pro 305 310 315 320 Asn Ala Gly Leu ThrMet Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys 325 330 335 Gly Pro Trp 4339 PRT Macaca sp. 4 Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys AsnTyr Arg Gly 1 5 10 15 Thr Met Ser Lys Thr Arg Thr Gly Ile Thr Cys GlnLys Trp Ser Ser 20 25 30 Thr Ser Pro His Arg Pro Thr Phe Ser Pro Ala ThrHis Pro Ser Glu 35 40 45 Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp AsnAsp Gly Gln Gly 50 55 60 Pro Trp Cys Tyr Thr Thr Asp Pro Glu Glu Arg PheAsp Tyr Cys Asp 65 70 75 80 Ile Pro Glu Cys Glu Asp Glu Cys Met His CysSer Gly Glu Asn Tyr 85 90 95 Asp Gly Lys Ile Ser Lys Thr Met Ser Gly LeuGlu Cys Gln Ala Trp 100 105 110 Asp Ser Gln Ser Pro His Ala His Gly TyrIle Pro Ser Lys Phe Pro 115 120 125 Asn Lys Asn Leu Lys Lys Asn Tyr CysArg Asn Pro Asp Gly Glu Pro 130 135 140 Arg Pro Trp Cys Phe Thr Thr AspPro Asn Lys Arg Trp Glu Leu Cys 145 150 155 160 Asp Ile Pro Arg Cys ThrThr Pro Pro Pro Ser Ser Gly Pro Thr Tyr 165 170 175 Gln Cys Leu Lys GlyThr Gly Glu Asn Tyr Arg Gly Asp Val Ala Val 180 185 190 Thr Val Ser GlyHis Thr Cys His Gly Trp Ser Ala Gln Thr Pro His 195 200 205 Thr His AsnArg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu 210 215 220 Asn TyrCys Arg Asn Pro Asp Gly Glu Lys Ala Pro Trp Cys Tyr Thr 225 230 235 240Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys Glu 245 250255 Ser Ser Pro Val Ser Thr Glu Pro Leu Asp Pro Thr Ala Pro Pro Glu 260265 270 Leu Thr Pro Val Val Gln Glu Cys Tyr His Gly Asp Gly Gln Ser Tyr275 280 285 Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln SerTrp 290 295 300 Ser Ser Met Thr Pro His Trp His Glu Lys Thr Pro Glu AsnPhe Pro 305 310 315 320 Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn ProAsp Ala Asp Lys 325 330 335 Gly Pro Trp 5 339 PRT Sus sp. 5 Ile Tyr LeuSer Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly 1 5 10 15 Thr ThrSer Lys Thr Lys Ser Gly Val Ile Cys Gln Lys Trp Ser Val 20 25 30 Ser SerPro His Ile Pro Lys Tyr Ser Pro Glu Lys Phe Pro Leu Ala 35 40 45 Gly LeuGlu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Lys Gly 50 55 60 Pro TrpCys Tyr Thr Thr Asp Pro Glu Thr Arg Phe Asp Tyr Cys Asp 65 70 75 80 IlePro Glu Cys Glu Asp Glu Cys Met His Cys Ser Gly Glu His Tyr 85 90 95 GluGly Lys Ile Ser Lys Thr Met Ser Gly Ile Glu Cys Gln Ser Trp 100 105 110Gly Ser Gln Ser Pro His Ala His Gly Tyr Leu Pro Ser Lys Phe Pro 115 120125 Asn Lys Asn Leu Lys Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro 130135 140 Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Phe Cys145 150 155 160 Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Thr Ser Gly ProThr Tyr 165 170 175 Gln Cys Leu Lys Gly Arg Gly Glu Asn Tyr Arg Gly ThrVal Ser Val 180 185 190 Thr Ala Ser Gly His Thr Cys Gln Arg Trp Ser AlaGln Ser Pro His 195 200 205 Lys His Asn Arg Thr Pro Glu Asn Phe Pro CysLys Asn Leu Glu Glu 210 215 220 Asn Tyr Cys Arg Asn Pro Asp Gly Glu ThrAla Pro Trp Cys Tyr Thr 225 230 235 240 Thr Asp Ser Glu Val Arg Trp AspTyr Cys Lys Ile Pro Ser Cys Gly 245 250 255 Ser Ser Thr Thr Ser Thr GluHis Leu Asp Ala Pro Val Pro Pro Glu 260 265 270 Gln Thr Pro Val Ala GlnAsp Cys Tyr Arg Gly Asn Gly Glu Ser Tyr 275 280 285 Arg Gly Thr Ser SerThr Thr Ile Thr Gly Arg Lys Cys Gln Ser Trp 290 295 300 Val Ser Met ThrPro His Arg His Glu Lys Thr Pro Gly Asn Phe Pro 305 310 315 320 Asn AlaGly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys 325 330 335 SerPro Trp 6 339 PRT Bos sp. 6 Ile Tyr Leu Leu Glu Cys Lys Thr Gly Asn GlyGln Thr Tyr Arg Gly 1 5 10 15 Thr Thr Ala Glu Thr Lys Ser Gly Val ThrCys Gln Lys Trp Ser Ala 20 25 30 Thr Ser Pro His Val Pro Lys Phe Ser ProGlu Lys Phe Pro Leu Ala 35 40 45 Gly Leu Glu Glu Asn Tyr Cys Arg Asn ProAsp Asn Asp Glu Asn Gly 50 55 60 Pro Trp Cys Tyr Thr Thr Asp Pro Asp LysArg Tyr Asp Tyr Cys Asp 65 70 75 80 Ile Pro Glu Cys Glu Asp Lys Cys MetHis Cys Ser Gly Glu Asn Tyr 85 90 95 Glu Gly Lys Ile Ala Lys Thr Met SerGly Arg Asp Cys Gln Ala Trp 100 105 110 Asp Ser Gln Ser Pro His Ala HisGly Tyr Ile Pro Ser Lys Phe Pro 115 120 125 Asn Lys Asn Leu Lys Met AsnTyr Cys Arg Asn Pro Asp Gly Glu Pro 130 135 140 Arg Pro Trp Cys Phe ThrThr Asp Pro Gln Lys Arg Trp Glu Phe Cys 145 150 155 160 Asp Ile Pro ArgCys Thr Thr Pro Pro Pro Ser Ser Gly Pro Lys Tyr 165 170 175 Gln Cys LeuLys Gly Thr Gly Lys Asn Tyr Gly Gly Thr Val Ala Val 180 185 190 Thr GluSer Gly His Thr Cys Gln Arg Trp Ser Glu Gln Thr Pro His 195 200 205 LysHis Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu 210 215 220Asn Tyr Cys Arg Asn Pro Asp Gly Glu Lys Ala Pro Trp Cys Tyr Thr 225 230235 240 Thr Asn Ser Glu Val Arg Trp Glu Tyr Cys Thr Ile Pro Ser Cys Glu245 250 255 Ser Ser Pro Leu Ser Thr Glu Arg Met Asp Val Pro Val Pro ProGlu 260 265 270 Gln Thr Pro Val Pro Gln Asp Cys Tyr His Gly Asn Gly GlnSer Tyr 275 280 285 Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly Arg Lys CysGln Ser Trp 290 295 300 Ser Ser Met Thr Pro His Arg His Leu Lys Thr ProGlu Asn Tyr Pro 305 310 315 320 Asn Ala Gly Leu Thr Met Asn Tyr Cys ArgAsn Pro Asp Ala Asp Lys 325 330 335 Ser Pro Trp 7 79 PRT Murinae gen.sp. misc_feature Kringle 1 7 Cys Lys Thr Gly Ile Gly Asn Gly Thr Arg GlyThr Met Ser Arg Thr 1 5 10 15 Lys Ser Gly Val Ala Cys Gln Lys Trp GlyAla Thr Phe Pro His Val 20 25 30 Pro Asn Tyr Ser Pro Ser Thr His Pro AsnGlu Gly Leu Glu Glu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Asn Asp Glu GlnGly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro Asp Lys Arg Tyr Asp Tyr CysAsn Ile Pro Glu Cys 65 70 75 8 79 PRT Homo sapiens misc_feature Kringle1 8 Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr 1 510 15 Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg 2025 30 Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn 3540 45 Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr 5055 60 Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys 65 7075 9 79 PRT Macaca sp. misc_feature Kringle 1 9 Cys Lys Thr Gly Asn GlyLys Asn Tyr Arg Gly Thr Met Ser Lys Thr 1 5 10 15 Arg Thr Gly Ile ThrCys Gln Lys Trp Ser Ser Thr Ser Pro His Arg 20 25 30 Pro Thr Phe Ser ProAla Thr His Pro Ser Glu Gly Leu Glu Glu Asn 35 40 45 Tyr Cys Arg Asn ProAsp Asn Asp Gly Gln Gly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro Glu GluArg Phe Asp Tyr Cys Asp Ile Pro Glu Cys 65 70 75 10 79 PRT Sus sp.misc_feature Kringle 1 10 Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg GlyThr Thr Ser Lys Thr 1 5 10 15 Lys Ser Gly Val Ile Cys Gln Lys Trp SerVal Ser Ser Pro His Ile 20 25 30 Pro Lys Tyr Ser Pro Glu Lys Phe Pro LeuAla Gly Leu Glu Glu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Asn Asp Glu LysGly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro Glu Thr Arg Phe Asp Tyr CysAsp Ile Pro Glu Cys 65 70 75 11 79 PRT Bos sp. misc_feature Kringle 1 11Cys Lys Thr Gly Asn Gly Gln Thr Tyr Arg Gly Thr Thr Ala Glu Thr 1 5 1015 Lys Ser Gly Val Thr Cys Gln Lys Trp Ser Ala Thr Ser Pro His Val 20 2530 Pro Lys Phe Ser Pro Glu Lys Phe Pro Leu Ala Gly Leu Glu Glu Asn 35 4045 Tyr Cys Arg Asn Pro Asp Asn Asp Glu Asn Gly Pro Trp Cys Tyr Thr 50 5560 Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asp Ile Pro Glu Cys 65 70 7512 78 PRT Murinae gen. sp. misc_feature Kringle 2 12 Cys Met Tyr Cys SerGly Glu Lys Tyr Glu Gly Lys Ile Ser Lys Thr 1 5 10 15 Met Ser Gly LeuAsp Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala 20 25 30 His Gly Tyr IlePro Ala Lys Phe Pro Ser Lys Asn Leu Lys Met Asn 35 40 45 Tyr Cys His AsnPro Asp Gly Glu Pro Arg Pro Trp Cys Phe Thr Thr 50 55 60 Asp Pro Thr LysArg Trp Glu Tyr Cys Asp Ile Pro Arg Cys 65 70 75 13 78 PRT Homo sapiensmisc_feature Kringle 2 13 Cys Met His Cys Ser Gly Glu Asn Tyr Asp GlyLys Ile Ser Lys Thr 1 5 10 15 Met Ser Gly Leu Glu Cys Gln Ala Trp AspSer Gln Ser Pro His Ala 20 25 30 His Gly Tyr Ile Pro Ser Lys Phe Pro AsnLys Asn Leu Lys Lys Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Arg Glu Leu ArgPro Trp Cys Phe Thr Thr 50 55 60 Asp Pro Asn Lys Arg Trp Glu Leu Cys AspIle Pro Arg Cys 65 70 75 14 78 PRT Macaca sp. misc_feature Kringle 2 14Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr 1 5 1015 Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala 20 2530 His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn 35 4045 Tyr Cys Arg Asn Pro Asp Gly Glu Pro Arg Pro Trp Cys Phe Thr Thr 50 5560 Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys 65 70 75 1578 PRT Sus sp. misc_feature Kringle 2 15 Cys Met His Cys Ser Gly Glu HisTyr Glu Gly Lys Ile Ser Lys Thr 1 5 10 15 Met Ser Gly Ile Glu Cys GlnSer Trp Gly Ser Gln Ser Pro His Ala 20 25 30 His Gly Tyr Leu Pro Ser LysPhe Pro Asn Lys Asn Leu Lys Met Asn 35 40 45 Tyr Cys Arg Asn Pro Asp GlyGlu Pro Arg Pro Trp Cys Phe Thr Thr 50 55 60 Asp Pro Asn Lys Arg Trp GluPhe Cys Asp Ile Pro Arg Cys 65 70 75 16 78 PRT Bos sp. misc_featureKringle 2 16 Cys Met His Cys Ser Gly Glu Asn Tyr Glu Gly Lys Ile Ala LysThr 1 5 10 15 Met Ser Gly Arg Asp Cys Gln Ala Trp Asp Ser Gln Ser ProHis Ala 20 25 30 His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu LysMet Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Gly Glu Pro Arg Pro Trp Cys PheThr Thr 50 55 60 Asp Pro Gln Lys Arg Trp Glu Phe Cys Asp Ile Pro Arg Cys65 70 75 17 78 PRT Murinae gen. sp. misc_feature Kringle 3 17 Cys LeuLys Gly Arg Gly Glu Asn Tyr Arg Gly Thr Val Ser Val Thr 1 5 10 15 ValSer Gly Lys Thr Cys Gln Arg Trp Ser Glu Gln Thr Pro His Arg 20 25 30 HisAsn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu Asn 35 40 45 TyrCys Arg Asn Pro Asp Gly Glu Thr Ala Pro Trp Cys Tyr Thr Thr 50 55 60 AspSer Gln Leu Arg Trp Glu Tyr Cys Glu Ile Pro Ser Cys 65 70 75 18 78 PRTHomo sapiens misc_feature Kringle 3 18 Cys Leu Lys Gly Thr Gly Glu AsnTyr Arg Gly Asn Val Ala Val Thr 1 5 10 15 Val Ser Gly His Thr Cys GlnHis Trp Ser Ala Gln Thr Pro His Thr 20 25 30 His Asn Arg Thr Pro Glu AsnPhe Pro Cys Lys Asn Leu Asp Glu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp GlyLys Arg Ala Pro Trp Cys His Thr Thr 50 55 60 Asn Ser Gln Val Arg Trp GluTyr Cys Lys Ile Pro Ser Cys 65 70 75 19 78 PRT Macaca sp. misc_featureKringle 3 19 Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asp Val Ala ValThr 1 5 10 15 Val Ser Gly His Thr Cys His Gly Trp Ser Ala Gln Thr ProHis Thr 20 25 30 His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu AspGlu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Gly Glu Lys Ala Pro Trp Cys TyrThr Thr 50 55 60 Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys65 70 75 20 78 PRT Sus sp. misc_feature Kringle 3 20 Cys Leu Lys Gly ArgGly Glu Asn Tyr Arg Gly Thr Val Ser Val Thr 1 5 10 15 Ala Ser Gly HisThr Cys Gln Arg Trp Ser Ala Gln Ser Pro His Lys 20 25 30 His Asn Arg ThrPro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu Asn 35 40 45 Tyr Cys Arg AsnPro Asp Gly Glu Thr Ala Pro Trp Cys Tyr Thr Thr 50 55 60 Asp Ser Glu ValArg Trp Asp Tyr Cys Lys Ile Pro Ser Cys 65 70 75 21 78 PRT Bos sp.misc_feature Kringle 3 21 Cys Leu Lys Gly Thr Gly Lys Asn Tyr Gly GlyThr Val Ala Val Thr 1 5 10 15 Glu Ser Gly His Thr Cys Gln Arg Trp SerGlu Gln Thr Pro His Lys 20 25 30 His Asn Arg Thr Pro Glu Asn Phe Pro CysLys Asn Leu Glu Glu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Gly Glu Lys AlaPro Trp Cys Tyr Thr Thr 50 55 60 Asn Ser Glu Val Arg Trp Glu Tyr Cys ThrIle Pro Ser Cys 65 70 75 22 78 PRT Murinae gen. sp. misc_feature Kringle4 22 Cys Tyr Gln Ser Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser Thr Thr 1 510 15 Ile Thr Gly Lys Lys Cys Gln Ser Trp Ala Ala Met Phe Pro His Arg 2025 30 His Ser Lys Thr Pro Glu Asn Phe Pro Asp Ala Gly Leu Glu Met Asn 3540 45 Tyr Cys Arg Asn Pro Asp Gly Asp Lys Gly Pro Trp Cys Tyr Thr Thr 5055 60 Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Arg Cys 65 70 7523 78 PRT Homo sapiens misc_feature Kringle 4 23 Cys Tyr His Gly Asp GlyGln Ser Tyr Arg Gly Thr Ser Ser Thr Thr 1 5 10 15 Thr Thr Gly Lys LysCys Gln Ser Trp Ser Ser Met Thr Pro His Arg 20 25 30 His Gln Lys Thr ProGlu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn 35 40 45 Tyr Cys Arg Asn ProAsp Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr 50 55 60 Asp Pro Ser Val ArgTrp Glu Tyr Cys Asn Leu Lys Lys Cys 65 70 75 24 168 PRT Murinae gen. sp.misc_feature Kringle 2-3 24 Cys Met Tyr Cys Ser Gly Glu Lys Tyr Glu GlyLys Ile Ser Lys Thr 1 5 10 15 Met Ser Gly Leu Asp Cys Gln Ala Trp AspSer Gln Ser Pro His Ala 20 25 30 His Gly Tyr Ile Pro Ala Lys Phe Pro SerLys Asn Leu Lys Met Asn 35 40 45 Tyr Cys His Asn Pro Asp Gly Glu Pro ArgPro Trp Cys Phe Thr Thr 50 55 60 Asp Pro Thr Lys Arg Trp Glu Tyr Cys AspIle Pro Arg Cys Thr Thr 65 70 75 80 Pro Pro Pro Pro Pro Ser Pro Thr TyrGln Cys Leu Lys Gly Arg Gly 85 90 95 Glu Asn Tyr Arg Gly Thr Val Ser ValThr Val Ser Gly Lys Thr Cys 100 105 110 Gln Arg Trp Ser Glu Gln Thr ProHis Arg His Asn Arg Thr Pro Glu 115 120 125 Asn Phe Pro Cys Lys Asn LeuGlu Glu Asn Tyr Cys Arg Asn Pro Asp 130 135 140 Gly Glu Thr Ala Pro TrpCys Tyr Thr Thr Asp Ser Gln Leu Arg Trp 145 150 155 160 Glu Tyr Cys GluIle Pro Ser Cys 165 25 168 PRT Homo sapiens misc_feature Kringle 2-3 25Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr 1 5 1015 Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala 20 2530 His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn 35 4045 Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr 50 5560 Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys Thr Thr 65 7075 80 Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly 8590 95 Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val Ser Gly His Thr Cys100 105 110 Gln His Trp Ser Ala Gln Thr Pro His Thr His Asn Arg Thr ProGlu 115 120 125 Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg AsnPro Asp 130 135 140 Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn Ser GlnVal Arg Trp 145 150 155 160 Glu Tyr Cys Lys Ile Pro Ser Cys 165 26 168PRT Macaca sp. misc_feature Kringle 2-3 26 Cys Met His Cys Ser Gly GluAsn Tyr Asp Gly Lys Ile Ser Lys Thr 1 5 10 15 Met Ser Gly Leu Glu CysGln Ala Trp Asp Ser Gln Ser Pro His Ala 20 25 30 His Gly Tyr Ile Pro SerLys Phe Pro Asn Lys Asn Leu Lys Lys Asn 35 40 45 Tyr Cys Arg Asn Pro AspGly Glu Pro Arg Pro Trp Cys Phe Thr Thr 50 55 60 Asp Pro Asn Lys Arg TrpGlu Leu Cys Asp Ile Pro Arg Cys Thr Thr 65 70 75 80 Pro Pro Pro Ser SerGly Pro Thr Tyr Gln Cys Leu Lys Gly Thr Gly 85 90 95 Glu Asn Tyr Arg GlyAsp Val Ala Val Thr Val Ser Gly His Thr Cys 100 105 110 His Gly Trp SerAla Gln Thr Pro His Thr His Asn Arg Thr Pro Glu 115 120 125 Asn Phe ProCys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn Pro Asp 130 135 140 Gly GluLys Ala Pro Trp Cys Tyr Thr Thr Asn Ser Gln Val Arg Trp 145 150 155 160Glu Tyr Cys Lys Ile Pro Ser Cys 165 27 168 PRT Sus sp. misc_featureKringle 2-3 27 Cys Met His Cys Ser Gly Glu His Tyr Glu Gly Lys Ile SerLys Thr 1 5 10 15 Met Ser Gly Ile Glu Cys Gln Ser Trp Gly Ser Gln SerPro His Ala 20 25 30 His Gly Tyr Leu Pro Ser Lys Phe Pro Asn Lys Asn LeuLys Met Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Gly Glu Pro Arg Pro Trp CysPhe Thr Thr 50 55 60 Asp Pro Asn Lys Arg Trp Glu Phe Cys Asp Ile Pro ArgCys Thr Thr 65 70 75 80 Pro Pro Pro Thr Ser Gly Pro Thr Tyr Gln Cys LeuLys Gly Arg Gly 85 90 95 Glu Asn Tyr Arg Gly Thr Val Ser Val Thr Ala SerGly His Thr Cys 100 105 110 Gln Arg Trp Ser Ala Gln Ser Pro His Lys HisAsn Arg Thr Pro Glu 115 120 125 Asn Phe Pro Cys Lys Asn Leu Glu Glu AsnTyr Cys Arg Asn Pro Asp 130 135 140 Gly Glu Thr Ala Pro Trp Cys Tyr ThrThr Asp Ser Glu Val Arg Trp 145 150 155 160 Asp Tyr Cys Lys Ile Pro SerCys 165 28 168 PRT Bos sp. misc_feature Kringle 2-3 28 Cys Met His CysSer Gly Glu Asn Tyr Glu Gly Lys Ile Ala Lys Thr 1 5 10 15 Met Ser GlyArg Asp Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala 20 25 30 His Gly TyrIle Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Met Asn 35 40 45 Tyr Cys ArgAsn Pro Asp Gly Glu Pro Arg Pro Trp Cys Phe Thr Thr 50 55 60 Asp Pro GlnLys Arg Trp Glu Phe Cys Asp Ile Pro Arg Cys Thr Thr 65 70 75 80 Pro ProPro Ser Ser Gly Pro Lys Tyr Gln Cys Leu Lys Gly Thr Gly 85 90 95 Lys AsnTyr Gly Gly Thr Val Ala Val Thr Glu Ser Gly His Thr Cys 100 105 110 GlnArg Trp Ser Glu Gln Thr Pro His Lys His Asn Arg Thr Pro Glu 115 120 125Asn Phe Pro Cys Lys Asn Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp 130 135140 Gly Glu Lys Ala Pro Trp Cys Tyr Thr Thr Asn Ser Glu Val Arg Trp 145150 155 160 Glu Tyr Cys Thr Ile Pro Ser Cys 165 29 250 PRT Murinae gen.sp. misc_feature Kringle 1-3 29 Cys Lys Thr Gly Ile Gly Asn Gly Tyr ArgGly Thr Met Ser Arg Thr 1 5 10 15 Lys Ser Gly Val Ala Cys Gln Lys TrpGly Ala Thr Phe Pro His Val 20 25 30 Pro Asn Tyr Ser Pro Ser Thr His ProAsn Glu Gly Leu Glu Glu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Asn Asp GluGln Gly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro Asp Lys Arg Tyr Asp TyrCys Asn Ile Pro Glu Cys Glu 65 70 75 80 Glu Glu Cys Met Tyr Cys Ser GlyGlu Lys Tyr Glu Gly Lys Ile Ser 85 90 95 Lys Thr Met Ser Gly Leu Asp CysGln Ala Trp Asp Ser Gln Ser Pro 100 105 110 His Ala His Gly Tyr Ile ProAla Lys Phe Pro Ser Lys Asn Leu Lys 115 120 125 Met Asn Tyr Cys His AsnPro Asp Gly Glu Pro Arg Pro Trp Cys Phe 130 135 140 Thr Thr Asp Pro ThrLys Arg Trp Glu Tyr Cys Asp Ile Pro Arg Cys 145 150 155 160 Thr Thr ProPro Pro Pro Pro Ser Pro Thr Tyr Gln Cys Leu Lys Gly 165 170 175 Arg GlyGlu Asn Tyr Arg Gly Thr Val Ser Val Thr Val Ser Gly Lys 180 185 190 ThrCys Gln Arg Trp Ser Glu Gln Thr Pro His Arg His Asn Arg Thr 195 200 205Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu Asn Tyr Cys Arg Asn 210 215220 Pro Asp Gly Glu Thr Ala Pro Trp Cys Tyr Thr Thr Asp Ser Gln Leu 225230 235 240 Arg Trp Glu Tyr Cys Glu Ile Pro Ser Cys 245 250 30 250 PRTHomo sapien misc_feature Kringle 1-3 30 Cys Lys Thr Gly Asn Gly Lys AsnTyr Arg Gly Thr Met Ser Lys Thr 1 5 10 15 Lys Asn Gly Ile Thr Cys GlnLys Trp Ser Ser Thr Ser Pro His Arg 20 25 30 Pro Arg Phe Ser Pro Ala ThrHis Pro Ser Glu Gly Leu Glu Glu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp AsnAsp Pro Gln Gly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro Glu Lys Arg TyrAsp Tyr Cys Asp Ile Leu Glu Cys Glu 65 70 75 80 Glu Glu Cys Met His CysSer Gly Glu Asn Tyr Asp Gly Lys Ile Ser 85 90 95 Lys Thr Met Ser Gly LeuGlu Cys Gln Ala Trp Asp Ser Gln Ser Pro 100 105 110 His Ala His Gly TyrIle Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys 115 120 125 Lys Asn Tyr CysArg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe 130 135 140 Thr Thr AspPro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys 145 150 155 160 ThrThr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly 165 170 175Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val Ser Gly His 180 185190 Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His Asn Arg Thr 195200 205 Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr Cys Arg Asn210 215 220 Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr Thr Asn Ser GlnVal 225 230 235 240 Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys 245 250 31250 PRT Macaca sp. misc_feature Kringle 1-3 31 Cys Lys Thr Gly Asn GlyLys Asn Tyr Arg Gly Thr Met Ser Lys Thr 1 5 10 15 Arg Thr Gly Ile ThrCys Gln Lys Trp Ser Ser Thr Ser Pro His Arg 20 25 30 Pro Thr Phe Ser ProAla Thr His Pro Ser Glu Gly Leu Glu Glu Asn 35 40 45 Tyr Cys Arg Asn ProAsp Asn Asp Gly Gln Gly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro Glu GluArg Phe Asp Tyr Cys Asp Ile Pro Glu Cys Glu 65 70 75 80 Asp Glu Cys MetHis Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser 85 90 95 Lys Thr Met SerGly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro 100 105 110 His Ala HisGly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys 115 120 125 Lys AsnTyr Cys Arg Asn Pro Asp Gly Glu Pro Arg Pro Trp Cys Phe 130 135 140 ThrThr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys 145 150 155160 Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly 165170 175 Thr Gly Glu Asn Tyr Arg Gly Asp Val Ala Val Thr Val Ser Gly His180 185 190 Thr Cys His Gly Trp Ser Ala Gln Thr Pro His Thr His Asn ArgThr 195 200 205 Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn Tyr CysArg Asn 210 215 220 Pro Asp Gly Glu Lys Ala Pro Trp Cys Tyr Thr Thr AsnSer Gln Val 225 230 235 240 Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys 245250 32 250 PRT Sus sp. misc_feature Kringle 1-3 32 Cys Lys Thr Gly AsnGly Lys Asn Tyr Arg Gly Thr Thr Ser Lys Thr 1 5 10 15 Lys Ser Gly ValIle Cys Gln Lys Trp Ser Val Ser Ser Pro His Ile 20 25 30 Pro Lys Tyr SerPro Glu Lys Phe Pro Leu Ala Gly Leu Glu Glu Asn 35 40 45 Tyr Cys Arg AsnPro Asp Asn Asp Glu Lys Gly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro GluThr Arg Phe Asp Tyr Cys Asp Ile Pro Glu Cys Glu 65 70 75 80 Asp Glu CysMet His Cys Ser Gly Glu His Tyr Glu Gly Lys Ile Ser 85 90 95 Lys Thr MetSer Gly Ile Glu Cys Gln Ser Trp Gly Ser Gln Ser Pro 100 105 110 His AlaHis Gly Tyr Leu Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys 115 120 125 MetAsn Tyr Cys Arg Asn Pro Asp Gly Glu Pro Arg Pro Trp Cys Phe 130 135 140Thr Thr Asp Pro Asn Lys Arg Trp Glu Phe Cys Asp Ile Pro Arg Cys 145 150155 160 Thr Thr Pro Pro Pro Thr Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly165 170 175 Arg Gly Glu Asn Tyr Arg Gly Thr Val Ser Val Thr Ala Ser GlyHis 180 185 190 Thr Cys Gln Arg Trp Ser Ala Gln Ser Pro His Lys His AsnArg Thr 195 200 205 Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu Asn TyrCys Arg Asn 210 215 220 Pro Asp Gly Glu Thr Ala Pro Trp Cys Tyr Thr ThrAsp Ser Glu Val 225 230 235 240 Arg Trp Asp Tyr Cys Lys Ile Pro Ser Cys245 250 33 250 PRT Bos sp. misc_feature Kringle 1-3 33 Cys Lys Thr GlyAsn Gly Gln Thr Tyr Arg Gly Thr Thr Ala Glu Thr 1 5 10 15 Lys Ser GlyVal Thr Cys Gln Lys Trp Ser Ala Thr Ser Pro His Val 20 25 30 Pro Lys PheSer Pro Glu Lys Phe Pro Leu Ala Gly Leu Glu Glu Asn 35 40 45 Tyr Cys ArgAsn Pro Asp Asn Asp Glu Asn Gly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp ProAsp Lys Arg Tyr Asp Tyr Cys Asp Ile Pro Glu Cys Glu 65 70 75 80 Asp LysCys Met His Cys Ser Gly Glu Asn Tyr Glu Gly Lys Ile Ala 85 90 95 Lys ThrMet Ser Gly Arg Asp Cys Gln Ala Trp Asp Ser Gln Ser Pro 100 105 110 HisAla His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys 115 120 125Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro Arg Pro Trp Cys Phe 130 135140 Thr Thr Asp Pro Gln Lys Arg Trp Glu Phe Cys Asp Ile Pro Arg Cys 145150 155 160 Thr Thr Pro Pro Pro Ser Ser Gly Pro Lys Tyr Gln Cys Leu LysGly 165 170 175 Thr Gly Lys Asn Tyr Gly Gly Thr Val Ala Val Thr Glu SerGly His 180 185 190 Thr Cys Gln Arg Trp Ser Glu Gln Thr Pro His Lys HisAsn Arg Thr 195 200 205 Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu AsnTyr Cys Arg Asn 210 215 220 Pro Asp Gly Glu Lys Ala Pro Trp Cys Tyr ThrThr Asn Ser Glu Val 225 230 235 240 Arg Trp Glu Tyr Cys Thr Ile Pro SerCys 245 250 34 160 PRT Murinae gen. sp. misc_feature Kringle 1-2 34 CysLys Thr Gly Ile Gly Asn Gly Tyr Arg Gly Thr Met Ser Arg Thr 1 5 10 15Lys Ser Gly Val Ala Cys Gln Lys Trp Gly Ala Thr Phe Pro His Val 20 25 30Pro Asn Tyr Ser Pro Ser Thr His Pro Asn Glu Gly Leu Glu Glu Asn 35 40 45Tyr Cys Arg Asn Pro Asp Asn Asp Glu Gln Gly Pro Trp Cys Tyr Thr 50 55 60Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asn Ile Pro Glu Cys Glu 65 70 7580 Glu Glu Cys Met Tyr Cys Ser Gly Glu Lys Tyr Glu Gly Lys Ile Ser 85 9095 Lys Thr Met Ser Gly Leu Asp Cys Gln Ala Trp Asp Ser Gln Ser Pro 100105 110 His Ala His Gly Tyr Ile Pro Ala Lys Phe Pro Ser Lys Asn Leu Lys115 120 125 Met Asn Tyr Cys His Asn Pro Asp Gly Glu Pro Arg Pro Trp CysPhe 130 135 140 Thr Thr Asp Pro Thr Lys Arg Trp Glu Tyr Cys Asp Ile ProArg Cys 145 150 155 160 35 160 PRT Homo sapiens misc_feature Kringle 1-235 Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr 1 510 15 Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg 2025 30 Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn 3540 45 Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr 5055 60 Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu 6570 75 80 Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser85 90 95 Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro100 105 110 His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn LeuLys 115 120 125 Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro TrpCys Phe 130 135 140 Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp IlePro Arg Cys 145 150 155 160 36 160 PRT Macaca sp. misc_feature Kringle1-2 36 Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr 15 10 15 Arg Thr Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg20 25 30 Pro Thr Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn35 40 45 Tyr Cys Arg Asn Pro Asp Asn Asp Gly Gln Gly Pro Trp Cys Tyr Thr50 55 60 Thr Asp Pro Glu Glu Arg Phe Asp Tyr Cys Asp Ile Pro Glu Cys Glu65 70 75 80 Asp Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys IleSer 85 90 95 Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln SerPro 100 105 110 His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys AsnLeu Lys 115 120 125 Lys Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro Arg ProTrp Cys Phe 130 135 140 Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys AspIle Pro Arg Cys 145 150 155 160 37 160 PRT Sus sp. misc_feature Kringle1-2 37 Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Thr Ser Lys Thr 15 10 15 Lys Ser Gly Val Ile Cys Gln Lys Trp Ser Val Ser Ser Pro His Ile20 25 30 Pro Lys Tyr Ser Pro Glu Lys Phe Pro Leu Ala Gly Leu Glu Glu Asn35 40 45 Tyr Cys Arg Asn Pro Asp Asn Asp Glu Lys Gly Pro Trp Cys Tyr Thr50 55 60 Thr Asp Pro Glu Thr Arg Phe Asp Tyr Cys Asp Ile Pro Glu Cys Glu65 70 75 80 Asp Glu Cys Met His Cys Ser Gly Glu His Tyr Glu Gly Lys IleSer 85 90 95 Lys Thr Met Ser Gly Ile Glu Cys Gln Ser Trp Gly Ser Gln SerPro 100 105 110 His Ala His Gly Tyr Leu Pro Ser Lys Phe Pro Asn Lys AsnLeu Lys 115 120 125 Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro Arg ProTrp Cys Phe 130 135 140 Thr Thr Asp Pro Asn Lys Arg Trp Glu Phe Cys AspIle Pro Arg Cys 145 150 155 160 38 160 PRT Bos sp. misc_feature Kringle1-2 38 Cys Lys Thr Gly Asn Gly Gln Thr Tyr Arg Gly Thr Thr Ala Glu Thr 15 10 15 Lys Ser Gly Val Thr Cys Gln Lys Trp Ser Ala Thr Ser Pro His Val20 25 30 Pro Lys Phe Ser Pro Glu Lys Phe Pro Leu Ala Gly Leu Glu Glu Asn35 40 45 Tyr Cys Arg Asn Pro Asp Asn Asp Glu Asn Gly Pro Trp Cys Tyr Thr50 55 60 Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asp Ile Pro Glu Cys Glu65 70 75 80 Asp Lys Cys Met His Cys Ser Gly Glu Asn Tyr Glu Gly Lys IleAla 85 90 95 Lys Thr Met Ser Gly Arg Asp Cys Gln Ala Trp Asp Ser Gln SerPro 100 105 110 His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys AsnLeu Lys 115 120 125 Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro Arg ProTrp Cys Phe 130 135 140 Thr Thr Asp Pro Gln Lys Arg Trp Glu Phe Cys AspIle Pro Arg Cys 145 150 155 160 39 352 PRT Murinae gen. sp. misc_featureKringle 1-4 39 Cys Lys Thr Gly Ile Gly Asn Gly Tyr Arg Gly Thr Met SerArg Thr 1 5 10 15 Lys Ser Gly Val Ala Cys Gln Lys Trp Gly Ala Thr PhePro His Val 20 25 30 Pro Asn Tyr Ser Pro Ser Thr His Pro Asn Glu Gly LeuGlu Glu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Asn Asp Glu Gln Gly Pro TrpCys Tyr Thr 50 55 60 Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asn Ile ProGlu Cys Glu 65 70 75 80 Glu Glu Cys Met Tyr Cys Ser Gly Glu Lys Tyr GluGly Lys Ile Ser 85 90 95 Lys Thr Met Ser Gly Leu Asp Cys Gln Ala Trp AspSer Gln Ser Pro 100 105 110 His Ala His Gly Tyr Ile Pro Ala Lys Phe ProSer Lys Asn Leu Lys 115 120 125 Met Asn Tyr Cys His Asn Pro Asp Gly GluPro Arg Pro Trp Cys Phe 130 135 140 Thr Thr Asp Pro Thr Lys Arg Trp GluTyr Cys Asp Ile Pro Arg Cys 145 150 155 160 Thr Thr Pro Pro Pro Pro ProSer Pro Thr Tyr Gln Cys Leu Lys Gly 165 170 175 Arg Gly Glu Asn Tyr ArgGly Thr Val Ser Val Thr Val Ser Gly Lys 180 185 190 Thr Cys Gln Arg TrpSer Glu Gln Thr Pro His Arg His Asn Arg Thr 195 200 205 Pro Glu Asn PhePro Cys Lys Asn Leu Glu Glu Asn Tyr Cys Arg Asn 210 215 220 Pro Asp GlyGlu Thr Ala Pro Trp Cys Tyr Thr Thr Asp Ser Gln Leu 225 230 235 240 ArgTrp Glu Tyr Cys Glu Ile Pro Ser Cys Glu Ser Ser Ala Ser Pro 245 250 255Asp Gln Ser Asp Ser Ser Val Pro Pro Glu Glu Gln Thr Pro Val Val 260 265270 Gln Glu Cys Tyr Gln Ser Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser 275280 285 Thr Thr Ile Thr Gly Lys Lys Cys Gln Ser Trp Ala Ala Met Phe Pro290 295 300 His Arg His Ser Lys Thr Pro Glu Asn Phe Pro Asp Ala Gly LeuGlu 305 310 315 320 Met Asn Tyr Cys Arg Asn Pro Asp Gly Asp Lys Gly ProTrp Cys Tyr 325 330 335 Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys AsnLeu Lys Arg Cys 340 345 350 40 352 PRT Homo sapiens misc_feature Kringle1-4 40 Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr 15 10 15 Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg20 25 30 Pro Arg Phe Ser Pro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn35 40 45 Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr50 55 60 Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu65 70 75 80 Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys IleSer 85 90 95 Lys Thr Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln SerPro 100 105 110 His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys AsnLeu Lys 115 120 125 Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg ProTrp Cys Phe 130 135 140 Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys AspIle Pro Arg Cys 145 150 155 160 Thr Thr Pro Pro Pro Ser Ser Gly Pro ThrTyr Gln Cys Leu Lys Gly 165 170 175 Thr Gly Glu Asn Tyr Arg Gly Asn ValAla Val Thr Val Ser Gly His 180 185 190 Thr Cys Gln His Trp Ser Ala GlnThr Pro His Thr His Asn Arg Thr 195 200 205 Pro Glu Asn Phe Pro Cys LysAsn Leu Asp Glu Asn Tyr Cys Arg Asn 210 215 220 Pro Asp Gly Lys Arg AlaPro Trp Cys His Thr Thr Asn Ser Gln Val 225 230 235 240 Arg Trp Glu TyrCys Lys Ile Pro Ser Cys Asp Ser Ser Pro Val Ser 245 250 255 Thr Glu GlnLeu Ala Pro Thr Ala Pro Pro Glu Leu Thr Pro Val Val 260 265 270 Gln AspCys Tyr His Gly Asp Gly Gln Ser Tyr Arg Gly Thr Ser Ser 275 280 285 ThrThr Thr Thr Gly Lys Lys Cys Gln Ser Trp Ser Ser Met Thr Pro 290 295 300His Arg His Gln Lys Thr Pro Glu Asn Tyr Pro Asn Ala Gly Leu Thr 305 310315 320 Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe325 330 335 Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys LysCys 340 345 350 41 378 PRT Murinae gen. sp. misc_feature Kringle 1-4BKLS41 Leu Phe Glu Lys Arg Val Tyr Leu Ser Glu Cys Lys Thr Gly Ile Gly 1 510 15 Asn Gly Tyr Arg Gly Thr Met Ser Arg Thr Lys Ser Gly Val Ala Cys 2025 30 Gln Lys Trp Gly Ala Thr Phe Pro His Val Pro Asn Tyr Ser Pro Ser 3540 45 Thr His Pro Asn Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp 5055 60 Asn Asp Glu Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg 6570 75 80 Tyr Asp Tyr Cys Asn Ile Pro Glu Cys Glu Glu Glu Cys Met Tyr Cys85 90 95 Ser Gly Glu Lys Tyr Glu Gly Lys Ile Ser Lys Thr Met Ser Gly Leu100 105 110 Asp Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala His Gly TyrIle 115 120 125 Pro Ala Lys Phe Pro Ser Lys Asn Leu Lys Met Asn Tyr CysHis Asn 130 135 140 Pro Asp Gly Glu Pro Arg Pro Trp Cys Phe Thr Thr AspPro Thr Lys 145 150 155 160 Arg Trp Glu Tyr Cys Asp Ile Pro Arg Cys ThrThr Pro Pro Pro Pro 165 170 175 Pro Ser Pro Thr Tyr Gln Cys Leu Lys GlyArg Gly Glu Asn Tyr Arg 180 185 190 Gly Thr Val Ser Val Thr Val Ser GlyLys Thr Cys Gln Arg Trp Ser 195 200 205 Glu Gln Thr Pro His Arg His AsnArg Thr Pro Glu Asn Phe Pro Cys 210 215 220 Lys Asn Leu Glu Glu Asn TyrCys Arg Asn Pro Asp Gly Glu Thr Ala 225 230 235 240 Pro Trp Cys Tyr ThrThr Asp Ser Gln Leu Arg Trp Glu Tyr Cys Glu 245 250 255 Ile Pro Ser CysGlu Ser Ser Ala Ser Pro Asp Gln Ser Asp Ser Ser 260 265 270 Val Pro ProGlu Glu Gln Thr Pro Val Val Gln Glu Cys Tyr Gln Ser 275 280 285 Asp GlyGln Ser Tyr Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly Lys 290 295 300 LysCys Gln Ser Trp Ala Ala Met Phe Pro His Arg His Ser Lys Thr 305 310 315320 Pro Glu Asn Phe Pro Asp Ala Gly Leu Glu Met Asn Tyr Cys Arg Asn 325330 335 Pro Asp Gly Asp Lys Gly Pro Trp Cys Tyr Thr Thr Asp Pro Ser Val340 345 350 Arg Trp Glu Tyr Cys Asn Leu Lys Arg Cys Ser Glu Thr Gly GlySer 355 360 365 Val Val Glu Leu Pro Thr Val Ser Gln Glu 370 375 42 368PRT Homo sapiens misc_feature Kringle 1-4 BKLS 42 Cys Lys Thr Gly AsnGly Lys Asn Tyr Arg Gly Thr Met Ser Lys Thr 1 5 10 15 Lys Asn Gly IleThr Cys Gln Lys Trp Ser Ser Thr Ser Pro His Arg 20 25 30 Pro Arg Phe SerPro Ala Thr His Pro Ser Glu Gly Leu Glu Glu Asn 35 40 45 Tyr Cys Arg AsnPro Asp Asn Asp Pro Gln Gly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro GluLys Arg Tyr Asp Tyr Cys Asp Ile Leu Glu Cys Glu 65 70 75 80 Glu Glu CysMet His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser 85 90 95 Lys Thr MetSer Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro 100 105 110 His AlaHis Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys 115 120 125 LysAsn Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe 130 135 140Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys 145 150155 160 Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr Tyr Gln Cys Leu Lys Gly165 170 175 Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala Val Thr Val Ser GlyHis 180 185 190 Thr Cys Gln His Trp Ser Ala Gln Thr Pro His Thr His AsnArg Thr 195 200 205 Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu Asn TyrCys Arg Asn 210 215 220 Pro Asp Gly Lys Arg Ala Pro Trp Cys His Thr ThrAsn Ser Gln Val 225 230 235 240 Arg Trp Glu Tyr Cys Lys Ile Pro Ser CysAsp Ser Ser Pro Val Ser 245 250 255 Thr Glu Gln Leu Ala Pro Thr Ala ProPro Glu Leu Thr Pro Val Val 260 265 270 Gln Asp Cys Tyr His Gly Asp GlyGln Ser Tyr Arg Gly Thr Ser Ser 275 280 285 Thr Thr Thr Thr Gly Lys LysCys Gln Ser Trp Ser Ser Met Thr Pro 290 295 300 His Arg His Gln Lys ThrPro Glu Asn Tyr Pro Asn Ala Gly Leu Thr 305 310 315 320 Met Asn Tyr CysArg Asn Pro Asp Ala Asp Lys Gly Pro Trp Cys Phe 325 330 335 Thr Thr AspPro Ser Val Arg Trp Glu Tyr Cys Asn Leu Lys Lys Cys 340 345 350 Ser GluThr Glu Ala Ser Val Val Ala Pro Pro Pro Val Val Leu Leu 355 360 365 4330 DNA Artificial Sequence PCR primer 43 atcgctcgag cgttatttgaaaagaaagtg 30 44 28 DNA Artificial Sequence PCR primer 44 atcggaattcaagcaggaca acaggcgg 28 45 28 DNA Artificial Sequence PCR primer 45atcgtacgta ttatttgaaa agaaagtg 28 46 459 PRT Murinae gen. sp.misc_feature Kringle 1-5 46 Glu Cys Lys Thr Gly Ile Gly Asn Gly Tyr ArgGly Thr Met Ser Arg 1 5 10 15 Thr Lys Ser Gly Val Ala Cys Gln Lys TrpGly Ala Thr Phe Pro His 20 25 30 Val Pro Asn Tyr Ser Pro Ser Thr His ProAsn Glu Gly Leu Glu Glu 35 40 45 Asn Tyr Cys Arg Asn Pro Asp Asn Asp GluGln Gly Pro Trp Cys Tyr 50 55 60 Thr Thr Asp Pro Asp Lys Arg Tyr Asp TyrCys Asn Ile Pro Glu Cys 65 70 75 80 Glu Glu Glu Cys Met Tyr Cys Ser GlyGlu Lys Tyr Glu Gly Lys Ile 85 90 95 Ser Lys Thr Met Ser Gly Leu Asp CysGln Ala Trp Asp Ser Gln Ser 100 105 110 Pro His Ala His Gly Tyr Ile ProAla Lys Phe Pro Ser Lys Asn Leu 115 120 125 Lys Met Asn Tyr Cys His AsnPro Asp Gly Glu Pro Arg Pro Trp Cys 130 135 140 Phe Thr Thr Asp Pro ThrLys Arg Trp Glu Tyr Cys Asp Ile Pro Arg 145 150 155 160 Cys Thr Thr ProPro Pro Pro Pro Ser Pro Thr Tyr Gln Cys Leu Lys 165 170 175 Gly Arg GlyGlu Asn Tyr Arg Gly Thr Val Ser Val Thr Val Ser Gly 180 185 190 Lys ThrCys Gln Arg Trp Ser Glu Gln Thr Pro His Arg His Asn Arg 195 200 205 ThrPro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu Asn Tyr Cys Arg 210 215 220Asn Pro Asp Gly Glu Thr Ala Pro Trp Cys Tyr Thr Thr Asp Ser Gln 225 230235 240 Leu Arg Trp Glu Tyr Cys Glu Ile Pro Ser Cys Glu Ser Ser Ala Ser245 250 255 Pro Asp Gln Ser Asp Ser Ser Val Pro Pro Glu Glu Gln Thr ProVal 260 265 270 Val Gln Glu Cys Tyr Gln Ser Asp Gly Gln Ser Tyr Arg GlyThr Ser 275 280 285 Ser Thr Thr Ile Thr Gly Lys Lys Cys Gln Ser Trp AlaAla Met Phe 290 295 300 Pro His Arg His Ser Lys Thr Pro Glu Asn Phe ProAsp Ala Gly Leu 305 310 315 320 Glu Met Asn Tyr Cys Arg Asn Pro Asp GlyAsp Lys Gly Pro Trp Cys 325 330 335 Tyr Thr Thr Asp Pro Ser Val Arg TrpGlu Tyr Cys Asn Leu Lys Arg 340 345 350 Cys Ser Glu Thr Gly Gly Ser ValVal Glu Leu Pro Thr Val Ser Gln 355 360 365 Glu Pro Ser Gly Pro Ser AspSer Glu Thr Asp Cys Met Tyr Gly Asn 370 375 380 Gly Lys Asp Tyr Arg GlyLys Thr Ala Val Thr Ala Ala Gly Thr Pro 385 390 395 400 Cys Gln Gly TrpAla Ala Gln Glu Pro His Arg His Ser Ile Phe Thr 405 410 415 Pro Gln ThrAsn Pro Arg Ala Asp Leu Glu Lys Asn Tyr Cys Arg Asn 420 425 430 Pro AspGly Asp Val Asn Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg 435 440 445 LysLeu Tyr Asp Tyr Cys Asp Ile Pro Leu Cys 450 455 47 80 PRT Murinae gen.sp. misc_feature Kringle 5 47 Cys Met Tyr Gly Asn Gly Lys Asp Tyr ArgGly Lys Thr Ala Val Thr 1 5 10 15 Ala Ala Gly Thr Pro Cys Gln Gly TrpAla Ala Gln Glu Pro His Arg 20 25 30 His Ser Ile Phe Thr Pro Gln Thr AsnPro Arg Ala Asp Leu Glu Lys 35 40 45 Asn Tyr Cys Arg Asn Pro Asp Gly AspVal Asn Gly Pro Trp Cys Tyr 50 55 60 Thr Thr Asn Pro Arg Lys Leu Tyr AspTyr Cys Asp Ile Pro Leu Cys 65 70 75 80 48 11 PRT Artificial SequenceSynthetic hemagglutinin epitope tag 48 Tyr Pro Tyr Asp Val Pro Asp TyrAla Ser Leu 1 5 10 49 79 PRT Homo sapiens 49 Cys Lys Thr Gly Asn Gly LysAsn Tyr Arg Gly Thr Met Ser Lys Thr 1 5 10 15 Lys Asn Gly Ile Thr CysGln Lys Trp Ser Ser Thr Ser Pro His Arg 20 25 30 Pro Arg Phe Ser Pro AlaThr His Pro Ser Glu Gly Leu Glu Glu Asn 35 40 45 Tyr Cys Arg Asn Pro AspAsn Asp Pro Gln Gly Pro Trp Cys Tyr Thr 50 55 60 Thr Asp Pro Glu Lys ArgTyr Asp Tyr Cys Asp Ile Leu Glu Cys 65 70 75 50 78 PRT Homo sapiens 50Cys Met His Cys Ser Gly Glu Asn Tyr Asp Gly Lys Ile Ser Lys Thr 1 5 1015 Met Ser Gly Leu Glu Cys Gln Ala Trp Asp Ser Gln Ser Pro His Ala 20 2530 His Gly Tyr Ile Pro Ser Lys Phe Pro Asn Lys Asn Leu Lys Lys Asn 35 4045 Tyr Cys Arg Asn Pro Asp Arg Glu Leu Arg Pro Trp Cys Phe Thr Thr 50 5560 Asp Pro Asn Lys Arg Trp Glu Leu Cys Asp Ile Pro Arg Cys 65 70 75 5178 PRT Homo sapiens 51 Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly AsnVal Ala Val Thr 1 5 10 15 Val Ser Gly His Thr Cys Gln His Trp Ser AlaGln Thr Pro His Thr 20 25 30 His Asn Arg Thr Pro Glu Asn Phe Pro Cys LysAsn Leu Asp Glu Asn 35 40 45 Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala ProTrp Cys His Thr Thr 50 55 60 Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys IlePro Ser Cys 65 70 75 52 78 PRT Homo sapiens 52 Cys Tyr His Gly Asp GlyGln Ser Tyr Arg Gly Thr Ser Ser Thr Thr 1 5 10 15 Thr Thr Gly Lys LysCys Gln Ser Trp Ser Ser Met Thr Pro His Arg 20 25 30 His Gln Lys Thr ProGlu Asn Tyr Pro Asn Ala Gly Leu Thr Met Asn 35 40 45 Tyr Cys Arg Asn ProAsp Ala Asp Lys Gly Pro Trp Cys Phe Thr Thr 50 55 60 Asp Pro Ser Val ArgTrp Glu Tyr Cys Asn Leu Lys Lys Cys 65 70 75

We claim:
 1. A method of inhibiting endothelial cell proliferationcomprising administering to an endothelial cell a proliferationinhibiting amount of a plasminogen fragment having an amino acidsequence substantially similar to the kringle 1-5 region of aplasminogen molecule.
 2. The method of claim 1, wherein the plasminogenfragment is derived from murine plasminogen, human plasminogen, Rhesusplasminogen, porcine plasminogen or bovine plasminogen.
 3. The method ofclaim 1, wherein the plasminogen fragment corresponds to approximatelyamino acids 98 to 560 of a plasminogen molecule.
 4. A method of treatinga mammal with an angiogenic-mediated disease comprising administering tothe mammal a treatment effective amount of a plasminogen fragment havingan amino acid sequence substantially similar to the kringle 1-5 regionof a plasminogen molecule.
 5. The method of claim 4, wherein theplasminogen fragment is derived from murine plasminogen, humanplasminogen, Rhesus plasminogen, porcine plasminogen or bovineplasminogen.
 6. The method of claim 4, wherein the plasminogen fragmentcorresponds to approximately amino acids 98 to 560 of a plasminogenmolecule.
 7. A therapeutic composition for inhibiting endothelial cellproliferation comprising a pharmaceutically acceptable excipient and aplasminogen fragment having an amino acid sequence substantially similarto the kringle 1-5 region of a plasminogen molecule.
 8. The compositionof claim 7, wherein the plasminogen fragment is derived from murineplasminogen, human plasminogen, Rhesus plasminogen, porcine plasminogenor bovine plasminogen.
 9. The composition of claim 7, wherein theplasminogen fragment corresponds to approximately amino acids 98 to 560of a plasminogen molecule.
 10. A composition comprising an isolatednucleotide sequence that codes for a plasminogen fragment having anamino acid sequence substantially similar to the kringle 1-5 region of aplasminogen molecule.
 11. The composition of claim 10, wherein theplasminogen fragment is derived from murine plasminogen, humanplasminogen, Rhesus plasminogen, porcine plasminogen or bovineplasminogen.
 12. The composition of claim 10, wherein the plasminogenfragment corresponds to approximately amino acids 98 to 560 of aplasminogen molecule.
 13. The composition of claim 12, furthercomprising a vector associated with the DNA sequence encoding theplasminogen fragment, wherein the vector is capable of expressing theplasminogen fragment when present in a cell.
 14. The composition ofclaim 13, further comprising a cell containing said vector.
 15. A methodof expressing an plasminogen fragment having an endothelial cellproliferation inhibiting activity and having an amino acid sequencesubstantially similar to the kringle 1-5 region of a plasminogenmolecule, comprising transfecting in a mammalian cell a vector, whereinthe vector contains a DNA sequence encoding said plasminogen fragment,and wherein the vector is capable of expressing angiostatin fragmentwhen present in the cell.